On-column viral inactivation methods

ABSTRACT

The present invention is directed to a method of inactivating virus that is present during production of a polypeptide of interest. In particular, the present invention is directed to a method of on-column virus inactivation using a low pH and high salt wash solution that effectively inactivates viruses with minimum recovery loss of the polypeptide.

REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The content of the electronically submitted sequence listing (Name:2159_4250002_SequenceListing_ST25; Size: 124,894 bytes; and Date ofCreation: Mar. 21, 2016) is herein incorporated by reference in itsentirety.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention is directed to a method of inactivating virus thatis present during production of a polypeptide of interest. Inparticular, the present invention is directed to a method of on-columnvirus inactivation using a low pH and high salt wash solution thateffectively inactivates viruses with minimum recovery loss of thepolypeptide.

Background Art

With the advent of recombinant protein technology, a protein of interestcan be produced using cultured cell lines engineered to express theprotein. The use of the desired recombinant protein for pharmaceuticalapplications is however generally contingent on the ability to reliablyrecover adequate levels of the protein from impurities such, as hostcell, proteins, cell culture additives, and viruses. Variouschromatography methods have been employed to remove the impurities andto recover the protein.

A number of methods for inactivating viruses based on differentmechanisms are known in the art. Each method however has its owndisadvantages, and may not be suitable or optimal for some proteinproducts. For example, when low pH is used to, inactivate viruses, ithas the potential to precipitate proteins, cause aggregation of theproduct, and/or alter the conformation of certain proteins which canlead to product loss. In addition, during the protein purificationprocess, the low pH virus inactivation step is typically performed afterthe protein of interest has been eluted from the chromatography columnand held in a tank or vessel, especially if significant product loss maybe caused by low pH wash. Adding an extra step in a tank or vessel toinactivate virus is a cause for inconvenience.

Therefore, there are needs to develop on-column viral inactivation stepsthat can effectively inactivate viruses and at the same time can improvethe product yield in a convenient manner.

BRIEF SUMMARY OF THE INVENTION

The present invention pertains to a method of inactivating virus that ispresent during production of a polypeptide of interest, comprising: (a)binding the polypeptide to a chromatography matrix, and (b) performing avirus inactivation step by washing the polypeptide bound chromatographymatrix with a wash solution at a pH of lower than about 4.0, wherein thewash solution comprises a sufficient concentration of salt tosubstantially reduce elution of the polypeptide during the virusinactivation step.

In certain embodiments, the chromatography matrix is an affinitychromatography matrix. In certain embodiments, the affinitychromatography matrix is a Protein A column. In certain embodiments, theProtein A column is selected from the group consisting of MABSELECT™,MABSELECT™ SuRe, MABSELECT™ SuRe LX, ESHMUNO® A, AMSPHERE™ JWT203,TOYOPEARL® AF-rProtein A-650F, PROSEP®-vA Ultra, PROSEP® Ultra Plus, andPROSEP®-vA High Capacity, and any combination thereof. In someembodiments, the Protein A ligand is immobilized on a matrix selectedfrom the group consisting of dextran based matrix, agarose based matrix,polystyrene based matrix, hydrophilic polyvinyl ethyl based matrix,rigid polymethacrylate based matrix, porous polymer based matrix,controlled pore glass based matrix, and any combination thereof.

In certain embodiments, the chromatography matrix is a mixed-modechromatography matrix. In certain embodiments the chromatography matrixis a mixed-mode anion-exchange chromatography matrix. In certainembodiments, the mixed-mode chromatography matrix is selected from thegroup consisting of CAPTO™ Adhere, CAPTO™ MMC, ESHMUNO® HCX, CAPTO™ MMCImpRes, CAPTO™ Blue, NUVIA™ cPrime, BLUE SEPHAROSE® Fast Flow, CAPTO™Adhere ImpRes, CHT™ Ceramic Hydroxyapatite, CFT™ Ceramic Fluoroapatite,and, any combinations thereof. In some embodiments, the mixed-modechromatography matrix is selected from the group consisting of dextranbased matrix, agarose based matrix, polystyrene based matrix, polyvinylethyl hydrophilic polymer based matrix, macroporous highly crosslinkedpolymer based matrix, hydroxyapatite ((Ca5(PO4)3OH)2) based matrix,fluoroapatite ((Ca5(PO4)3F)2) based matrix, and any combinationsthereof.

In certain embodiments, the polypeptide of interest is aCH2/CH3-containing polypeptide. In certain embodiments, theCH2/CH3-containing polypeptide is an antibody or an antibody fragment.In one embodiment, the antibody is a monoclonal antibody.

In certain embodiments, the polypeptide of interest comprises a clottingfactor. In certain embodiments, the polypeptide of interest is FIX-Fc,FVIII-Fc, or FVII-Fc. In certain embodiments, the polypeptide is amonomer-dimer hybrid. In certain embodiments, the polypeptide furthercomprises a heterologous moiety. In one embodiment, the heterologousmoiety is selected from the group consisting of albumin, albumin-bindingpolypeptide, Fc, PAS, the C-terminal peptide (CTP) of the β subunit ofhuman chorionic gonadotropin, polyethylene glycol (PEG), hydroxyethylstarch (HES), albumin-binding small molecules, and any combinationsthereof.

In certain embodiments, the polypeptide of interest is recombinantlyproduced in a cell culture. In certain embodiments, the cell culture isa human cell culture. In one embodiment, the human cell culture, isHuman Embryonic Kidney (HEK) 293 cell.

In certain embodiments, the polypeptide of interest is harvested afterrecombinant production in the cell culture. In certain embodiments thepolypeptide is bound to the chromatography matrix at a pH from about 6.0to about 8.0.

In certain embodiments, the elution of the polypeptide during the virusinactivation step is reduced to less than 30%. In certain embodiments,the elution of the polypeptide during the virus inactivation step isreduced to less than 25%, less than 20%, less than 15%, less than 10%,or less than 5%.

In certain embodiments, the pH of the wash solution is about 2.5 toabout 4.0. In other embodiments, the pH of the wash solution is about2.5 to about 3.0, about 3.0 to about 3.5, or about 3.5 to about 4.0. Incertain embodiments, the pH of the wash solution is about 2.5, about2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.2,about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about3.9, or about 4.0.

In certain embodiments, the concentration of the salt in the washsolution is greater than about 0.5 M. In certain embodiments, theconcentration of the salt is about 0.5 M to about 1.0 M, about 1.0 M toabout 1.5 M, about 1.5 M to about 2.0 M, about 2.0 M to about 2.5 M,about 2.5 M to about 3.0 M, about 3.0 M to about 3.5 M, or about 3.5 Mto about 4 M.

In certain embodiments, the salt in the wash solution is a sodium salt,a potassium salt, or an ammonium salt.

In certain embodiments, the wash solution further comprises one or morecomponents selected from the group consisting of a polymer, an organicsolvent, a detergent, and arginine or an arginine derivative.

In certain embodiments, the method comprises more than onevirus-inactivation step, wherein identical or different wash solutionscan be used. In certain embodiments, at least one of the wash solutionscomprises arginine, an arginine derivative, or a mixture thereof. Incertain embodiments, at least one of the wash solutions comprises adetergent.

In certain embodiments, the method further comprises eluting thepolypeptide from the chromatography matrix with an elution solution. Incertain embodiments, at least, about 70% of the polypeptide is recoveredin the elution solution. In certain embodiments, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, or at leastabout 95% of the polypeptide is recovered in the elution solution.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1. The chromatogram showing, the separation of proteins in aProtein A chromatography column using a wash solution containing 2 Mammonium sulfate at pH 3.5. UV₂₈₀ indicates protein concentration in thecollected fractions.

FIG. 2. The chromatogram showing the separation of proteins in a ProteinA chromatography column using a wash solution containing 1 M arginine atpH 4.7. UV₂₈₀ indicates protein concentration in the collectedfractions.

FIG. 3. The chromatogram showing the separation of proteins in a ProteinA chromatography column using a wash solution containing 4×CMC (or 0.18%w/w) LDAO. UV₂₈₀ indicates protein concentration in the collectedfractions.

FIG. 4. The chromatogram showing the separation of proteins in a ProteinA chromatography column using a wash solution containing 2 M NaCl and20% PEG at pH 3.0. UV₂₈₀ indicates protein concentration in thecollected fractions.

FIG. 5. The chromatogram showing the separation of proteins in a ProteinA chromatography column using a wash solution containing 2 M NaCl and 2%ethanol at pH 3.0. UV₂₈₀ indicates protein concentration in thecollected fractions.

FIG. 6. The chromatogram showing the separation of proteins in a ProteinA chromatography column using a wash solution containing 2 M ammoniumsulfate and 2% ethanol at pH 3.0. UV₂₈₀ indicates protein concentrationin the collected fractions.

FIG. 7. The chromatogram showing the separation of proteins in a ProteinA chromatography column using a wash solution containing 2 M ammoniumsulfate and 2% acetone at pH 3.0. UV₂₈₀ indicates protein concentrationin the collected fractions.

FIG. 8. The chromatogram showing the separation of proteins in a ProteinA chromatography column using a wash solution containing 2 M ammoniumsulfate at pH 3.0. UV₂₈₀ indicates protein concentration in thecollected fractions.

FIG. 9. The chromatogram showing the separation of proteins in a ProteinA chromatography column using a wash solution containing 2 M ammoniumsulfate and 2% TRITON™ X-100 at pH 3.0. UV₂₈₀ indicates proteinconcentration in the collected fractions.

FIG. 10. The chromatogram showing the separation of proteins in aProtein A chromatography column using a wash solution containing 2 MNaCl at pH 3.0. UV₂₈₀ indicates protein concentration in the collectedfractions.

FIG. 11. The chromatogram showing the separation of proteins in amixed-mode anion-exchange chromatography column using a wash solutioncontaining 2 M ammonium sulfate at, pH 3.5 and 4.0. UV₂₈₀ indicatesprotein concentration in the collected fractions.

FIG. 12. The concentration of ammonium sulfate was reduced from 2 M tozero over a 9 CV gradient at pH 3.0 to determine the minimumconcentration of ammonium sulfate required to keep the antibody bound tothe protein A resin at low pH values. At least 1700 mM ammonium sulfateis required to keep the antibody bound to the resin at low pH.

FIG. 13. Protein concentration, conductivity and pH versus columnvolumes using a pH 3.0, 2 M ammonium sulfate, 100 mM glycine wash. Thehigh level of ammonium sulfate prevented the low pH elution of theantibody, potentially enabling on-column viral inactivation.

FIG. 14. Protein concentration, conductivity and pH versus columnvolumes using a pH 3.5, 2 M ammonium sulfate, 100 mM citrate wash tokeep the antibody bound to the mixed mode anion exchange resin (CaptoAdhere) resin at low pH.

FIG. 15. Protein concentration, conductivity and pH versus columnvolumes using a pH 8.0, 2 M ammonium sulfate, 50 mM phosphate wash tokeep the antibody bound to the mixed mode cation exchange resin (CaptoMMC) resin at high pH.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

Throughout this disclosure, the term “a” or “an” entity refers to one ormore of that entity for example, “a polypeptide,” is understood torepresent one or more polypeptides. As such, the terms “a” (or “an”),“one or more,” and “at least one” can be used interchangeably herein.

Furthermore, “and/or” where used herein is to be taken as specificdisclosure of each of the two specified features or components with orwithout the other. Thus, the term “and/or” as used in a phrase such as“A and/or B” herein is intended to include “A and B,” “A or B,” “A”(alone), and “B” (alone). Likewise, the term “and/or” as used in aphrase such as “A, B, and/or C” is intended to encompass each of thefollowing aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; Aand C; A and B; B and C; A (alone); B (alone); and C (alone).

As used herein, the term “about” allows for the degree of variationinherent in the methods and in the instrumentation used for measurementor quantitation. For example, depending on the level of precision of theinstrumentation used, standard error based on the number of samplesmeasured, and rounding error, the term “about” includes, withoutlimitation, ±10%.

It is understood that wherever aspects are described herein with thelanguage “comprising,” otherwise analogous aspects described in terms of“consisting of and/or” “consisting essentially of” are also provided.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure is related. For example, the ConciseDictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed.,2002, ICRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed.,1999, Academic Press; and the Oxford Dictionary Of Biochemistry AndMolecular Biology, Revised, 2000, Oxford University Press, provide oneof skill with a general dictionary of many of the terms used in thisdisclosure.

Units, prefixes, and symbols are denoted in their Systéme Internationalde Unites (SI) accepted form. Numeric ranges are inclusive of thenumbers defining the range. Unless otherwise indicated, amino acidsequences are written left to right in amino to carboxy orientation. Theheadings provided herein are not limitations of the various aspects ofthe disclosure, which can be had by reference to the specification as awhole. Accordingly, the terms defined immediately below are more fullydefined by reference to the specification in its entirety.

The term “polypeptide” as used herein refers to a sequential chain ofamino acids linked together via peptide bonds. The term is used to referto an amino acid chain of any length, but one of ordinary skill in theart will understand that the term is not limited to lengthy chains andcan refer to a minimal chain comprising two amino acids linked togethervia a peptide bond. If a single polypeptide is the discrete functioningunit and does require permanent physical association with otherpolypeptides in order to form the discrete, functioning unit, the terms“polypeptide” and “protein” as used herein are used interchangeably. Ifdiscrete functional unit is comprised of more than one polypeptide thatphysically associate with one another, the term “protein” as used hereinrefers to the multiple polypeptides that are physically coupled andfunction together as the discrete unit. Thus, as used herein, a“peptide,” a “peptide fragment,” a “protein,” an “amino acid chain,” an“amino acid sequence,” or any other term used to refer to a chain orchains of two or more amino acids, are generically included in thedefinition of a “polypeptide,” even, though each of these terms can havea more specific meaning. The term “polypeptide” can be used instead of,or interchangeably with any of these terms. The term further includespolypeptides which have undergone post-translational or post-synthesismodifications, for example, glycosylation, acetylation, phosphorylation,amidation, derivatization by known protecting/blocking groups,proteolytic cleavage, or modification by non-naturally occurring aminoacids.

“Recombinantly expressed polypeptide” and “recombinant polypeptide” asused herein refer to a polypeptide expressed from a host cell that hasbeen genetically engineered to express that polypeptide. Therecombinantly expressed polypeptide can be identical or similar topolypeptides that are normally expressed in the host cell. Therecombinantly expressed polypeptide can also be foreign to the hostcell, i.e., heterologous to peptides normally expressed in the hostcell. Alternatively, the recombinantly expressed polypeptide can bechimeric in that portions of the polypeptide contain amino acidsequences that are identical or similar to polypeptides normallyexpressed in the host cell, while other portions are foreign to the hostcell. Host cells include, but are not limited to, prokaryotic cells,eukaryotic cells, plant cells, yeast cells, animal cells, insect cells,avian cells, and mammalian cells. As used herein, the terms“recombinantly expressed polypeptide” and “recombinant polypeptide” alsoencompasses an antibody produced by, a hybridoma.

The term “expression” or “expresses” are used herein to refer totranscription and translation occurring within a host cell. The level ofexpression of a product gene in a host cell can be determined on thebasis of either the amount of corresponding mRNA that is present in thecell or the amount of the protein encoded by the product, gene that isproduced by the cell. For example, mRNA transcribed from a product geneis desirably quantitated by northern hybridization. Sambrook et al.,Molecular Cloning: A Laboratory Manual, pp. 7.3-7.57 (Cold Spring HarborLaboratory Press, 1989). Protein encoded by a product gene can bequantitated either by assaying for the biological activity of theprotein or by employing assays that are independent of such activity,such as western blotting, ELISA, forteBIO, Bradford assay, absorbance at280 nm, or radioimmunoassay using antibodies that are capable ofreacting with the protein. Sambrook et al., Molecular Cloning: ALaboratory Manual, pp. 18.1-18.88 (Cold Spring Harbor Laboratory Press,1989).

The term “solution” refers to a mixture of one or more liquids(solvents) with one or more solids (solutes), such as a salt, a polymer,or a polypeptide. As used herein, a solution includes a buffer solution.

A “buffer” is a solution that resists changes in pH by the action of itsacid-base conjugate components. Various buffers which can be employeddepending, for example, on the desired pH of the buffer are described inBuffers. A Guide for the Preparation and Use of Buffers in BiologicalSystems, Gueffroy, D., ed. Calbiochem Corporation (1975). Many buffersare known in the art for use in buffer solutions and include, but arenot limited to histidine, citrate, phosphate, succinate,tris(hydroxymethyl)aminomethane (Tris), acetate, glycine, aconitate,maleate, phthalate, cacodylate, barbitol, 2-(N-morpholino)ethanesulfonicacid (MES), bis(2-hydroxyethyl)imino-tris-(hydroxymethyl)methane(Bistris), N-(2-Acetamido)iminodiacetic acid (ADA),piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES),1,3-bis[tris(hydroxymethyl)-methylamino]propane (Bistrispropane),N-(Acetamido)-2-aminoethanesulfonic acid (ACES),3-(N-morpholino)propanesulfonic acid (MOPS),N,N′-bis(2-hydroxyethyl)-2-amino-ethanesulfonic acid (BES),N-tris(hydroxymethyl)methyl-2-amino-ethanesulfonic acid (TES),N-2-hydroxyethylpiperazine-N′-ethanesulfonic acid (HEPES),N-2-hydroxyethylpiperazine-N′-propanesulfonic acid (HEPPS),N-tris(hydroxymethyl)methylglycine (Tricine),N,N-bis(2-hydroxyethyl)glycine (Bicine), glycylglycine,N-tris(hydroxymethyl)methyl-3-amino-propanesulfonic acid (TAPS),1,3-bis[tris(hydroxymethyl)-methylamino]propane (Bistrispropane), aswell as combinations of these.

The term “loading buffer” refers to the buffer, in which the polypeptidebeing purified is applied to a purification device, e.g., achromatography column or a filter cartridge. Typically, the loadingbuffer is selected so that separation of the polypeptide of interestfrom unwanted impurities can be accomplished.

The terms “wash solution” and “wash buffer” are used interchangeablyherein and refer to the buffer used to remove contaminant(s), such asprocess-related impurities, from the polypeptide-bound purificationdevice (e.g., a chromatography matrix) without removing significantamounts of the polypeptide of interest. The wash solution can comprise asalt, a detergent, a solvent, a polymer, or any combinations thereof.

The terms “elution solution” and “elution buffer” are usedinterchangeably herein and refer to the buffer, which is typically usedto remove (elute) the polypeptide of interest from the purificationdevice (e.g., a chromatographic column or filter cartridge) to which itwas applied earlier. Typically, the elution solution is selected so thatseparation of the polypeptide of interest from unwanted impurities canbe accomplished. Often, the concentration of a particular ingredient,such as a particular salt (e.g. NaCl) in the elution elution is variedduring the elution procedure (gradient). The gradient can be continuousor stepwise (interrupted by hold periods). In certain embodiments, lowpH, such as a pH value below 4.5, is used in an elution solution.

The term “chromatography” refers to the process by which a solute ofinterest, typically a polypeptide, in a mixture is separated from othersolutes in a mixture as a result of differences in rates at which theindividual solutes of the mixture migrate through a stationary mediumunder the influence of a moving phase, or in bind and elute processes.The chromatography steps of the present invention can employ any type ofchromatographic method. For example, such methods include withoutlimitation: gas chromatography, liquid chromatography (e.g., highperformance liquid chromatography); affinity chromatography (such asProtein-A or antibody-antigen affinity chromatography); supercriticalfluid chromatography; ion exchange chromatography (such as anion orcation exchange chromatography); size-exclusion chromatography; reversedphase chromatography; two-dimensional chromatography; simulated movingbed chromatography, pyrolysis gas chromatography, fast protein (FPLC)chromatography; countercurrent chromatography; chiral chromatography;aqueous normal phase (ANP) chromatography; mixed mode chromatography;and, pseudo-affinity chromatography.

Any or all chromatographic steps of the invention can be carried out byany mechanical means. Chromatography can be carried out in a column. Thecolumn can be run with or without pressure and from top to bottom orbottom to top. The direction of the flow of fluid in the column can bereversed during the chromatography process. Chromatography can also becarried out using a batch process in which the solid support isseparated from the liquid used to load, wash, and elute the sample byany suitable means, including gravity, centrifugation, or filtration.Chromatography can also be carried out by contacting the sample with afilter that absorbs or retains some molecules in the sample morestrongly than others.

The term “affinity chromatography” refers to a protein separationtechnique in which a polypeptide of interest is reversibly andspecifically bound to a biospecific ligand. Preferably, the biospecificligand is covalently attached to a chromatographic solid phase materialand is accessible to the polypeptide of interest in solution as thesolution contacts the chromatographic solid phase material. Thepolypeptide of interest (e.g., antibody, enzyme, or receptor protein)retains its specific binding affinity for the biospecific ligand(antigen, substrate, cofactor, or hormone, for example) during thechromatographic steps, while other solutes and/or proteins in themixture do not bind appreciably or specifically to the ligand. Bindingof the polypeptide of interest to the immobilized ligand allowscontaminating proteins or protein impurities to be passed through thechromatographic medium while the protein of interest remainsspecifically bound to the immobilized ligand on the solid phasematerial. The specifically bound polypeptide of interest is then removedin active form from the immobilized ligand with low pH, high pH, high sat, competing ligand, and the like, and passed through thechromatographic column with the elution buffer, free of thecontaminating proteins or protein impurities that were earlier allowedto pass through the column. Any component can be used as a ligand forpurifying its respective specific binding protein, e.g. antibody.

The terms “Protein A” and “ProA” are used interchangeably herein andencompasses Protein A recovered from a native source thereof, Protein Aproduced synthetically (e.g. by peptide synthesis or by recombinanttechniques), and variants thereof which retain the ability to bindproteins which have a CH2/CH3 region, such as an Fc region. Protein Acan be purchased commercially, for example, from Repligen, Pharmacia andFermatech. Protein A is generally immobilized on a solid phase supportmaterial. The term “ProA” also refers to an affinity chromatographyresin or column containing chromatographic solid support matrix to whichis covalently attached Protein A.

In practice, Protein A chromatography involves using Protein Aimmobilized to a solid support. Protein G and Protein LG can also beused for affinity chromatography. The solid support is a non-aqueousmatrix onto which Protein A adheres. Such supports include agarose,sepharose, glass, silica, polystyrene, collodion charcoal, sand, and anyother suitable material. Such materials are well known in the art. Anysuitable method can be used to affix the second protein to the solidsupport. Methods for affixing proteins to suitable solid supports arewell known in the art. Such solid supports, with and without immobilizedProtein A, are readily available from many commercial sources includingsuch as Vector Laboratory (Burlingame, Calif.), Santa Cruz Biotechnology(Santa Cruz, Calif.), BioRad (Hercules, Calif.), Amersham Biosciences(part of GE Healthcare, Uppsala, Sweden) and Millipore (Billerica,Mass.). Protein A immobilized to a pore glass matrix is commerciallyavailable as PROSEP®-A (Millipore). The solid phase can also be anagarose-based matrix. Protein A immobilized on an agarose matrix iscommercially available as MABSELECT™ (GE Healthcare, Uppsala, Sweden).

The term “mixed-mode chromatography” refers to a purification processusing, mixed mode adsorbents which provide multiple modes ofinteraction, such as hydrophobic, cation exchange, anion exchange, andhydrogen bonding interaction between the polypeptide of interest and theadsorbent ligands. A mixed-mode anion exchange resin is, one that hasboth anion exchange groups and hydrophobic groups on the ligand.Commercially available mixed mode chromatography resins include, but arenot limited to, CAPTO™ MMC, CAPTO™ MMC ImpRes, CAPTO™ Blue, BLUESEPHAROSE™ 6 Fast Flow, CAPTO™ Adhere, and CAPTO™ Adhere ImpRes from GEHealthcare Life Sciences, or ESHMUNO® HCX from EMD Millipore, or NUVIA™cPrime, CHT™ Ceramic Hydroxyapatite, and CFT™ Ceramic Fluoroapatite fromBio-Rad.

The terms “anion exchange resin,” “anion exchange adsorbent,” or “anionexchange matrix” are used herein to refer to a solid phase which ispositively charged, e.g. having one or more positively charged ligands,such as quaternary amino groups, attached thereto. Commerciallyavailable anion exchange resins include DEAE SEPHAROSE™ Fast Flow, QSEPHAROSE™ Fast Flow, Q SEPHAROSE™ High Performance, Q SEPHAROSE™ XL,CAPTO™ DEAE, CAPTO™ Q, and CAPTO™ Q ImpRes from GE Healthcare LifeSciences, or FRACTOGEL® EMD TMAE HiCap, FRACTOGEL® EMD DEAE, andESHMUNO® Q from EMD Millipore, or UNOSPHERE™ Q and NUVIA™ Q fromBio-Rad.

The terms “cation exchange resin,” “cation exchange adsorbent,” or“cation exchange matrix” refer to a solid phase which is negativelycharged, and which thus has free cations for exchange with cations in anaqueous solution passed over or through the solid phase. A negativelycharged ligand attached to the solid phase to form the cation exchangeresin can, e.g., be a carboxylate or sulfonate. Commercially availablecation exchange resins include carboxy-methyl-cellulose, sulphopropyl(SP) immobilized on agarose (e.g. SP SEPHAROSE™ XL, SP SEPHAROSE™ FastFlow, SP SEPHAROSE™ High Performance, CM SEPHAROSE™ Fast Flow, CMSEPHAROSE™ High Performance, CAPTO™ S, and CAPTO™ SP ImpRes from GEHealthcare Life Sciences, or FRACTOGEL® EMD SE HiCap, FRACTOGEL® EMDSO³⁻, FRACTOGEL® EMD COO⁻, ESHMUNO® S, and ESHMUNO® CPX from EMDMillipore, or UNOSPHERE™ S and NUVIA™ S from Bio-Rad).

As used herein, the terms “substantially reduce the elution of thepolypeptide” or “substantial reduction of the polypeptide elution” areintended to can that less than 30% of the target polypeptide is elutedfrom the chromatography matrix in a low pH and high salt wash solution.In one embodiment, less than 25%, less than 20%, less than 15%, lessthan 10%, or less than 5% of the target polypeptide is eluted from thechromatography matrix in a low pH high salt wash solution.

As used herein, the terms “percent recovery” and “percent purity,” areintended to mean the recovery or purity achieved when a target compound(e.g., a protein) is conveyed through a purification step or procedure,compared to the quantity or purity of the target compound in the sampleprior to the purification step or procedure. Achieving an increase inpercent purity entails obtaining a product with reduced levels ofcontaminants (in proportion to the target compound) when a sample iscompared before and after a purification step or procedure. Preferredpercentages within the meaning of percent recovery and percent purity asdefined above include, without limitation, at least about 50%, at leastabout 60%, at least about 70%, at least about 80%, at least about 90%,at least about 95%, at least about 98%, and at least about 99%.

Methods for the determination of yield or purity of a polypeptide areknown to those of skill in the art. Yield or purity of a polypeptide canbe determined by any suitable, art-recognized method of analysis (e.g.,band intensity on a silver stained gel, polyacrylamide gelelectrophoresis, ELISA, HPLC and the like). An exemplary method issize-exclusion chromatography (SEC) high-performance liquidchromatography (HPLC), described herein below. Purity can be determinedusing relative “area under the curve” (AUC) values, which can typicallybe obtained for peaks in a chromatogram, such as an HPLC chromatogram.Optionally, purities are determined by chromatographic or other meansusing a standard curve generated using a reference material of knownpurity. Purity can also be determined on a weight-by-weight basis.

As used herein, the term “inactivate” or other forms of this word (e.g.,inactivation, inactivated, inactivates, etc.) when used in reference toviruses is intended to indicate not only complete virus inactivation(i.e., no detectable infectious virus) but also the detectable reducingor reduction of infectious virus titers (i.e., lowering or loweredlevels of detectable infectious virus). Thus, the reducing or reductionof infectious virus titers is included within the meaning of “virusinactivation” (and other forms of this term) whether or not suchreducing or reduction is explicitly stated herein. Quantificationmethods for viral inactivation are well known in the art. Methods suchas plaque assays can be used. Plaque assays determine the number ofplaque forming units (pfu) in a virus sample, assuming that each plaqueformed is representative of one infective virus particle or TCID50assays, where an endpoint dilution assay quantifies the amount of virusrequired to kill 50% of infected hosts or to produce a cytopathic effectin 50% of inoculated tissue culture cells.

The term “polymer” refers to a molecule formed by covalent linkage oftwo or more monomers, where the monomers are not amino acids.Non-limiting examples of polymers include polyethyl glycol, polypropylglycol, and copolymers of ethylene and propylene glycol.

The term “detergent” refers to nonionic or zwitterionic surfactants suchas polysorbates (e.g. polysorbates 20 or 80); poloxamers (e.g. poloxamer188); octylphenol ethylene oxide condensate (also known as Octoxynol-9,t-octylphenoxypolyethoxyethanol, TRITON™, or TRITON™ X-100);3[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS);3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate(CHAPSO); sodium dodecyl sulfate (SDS), sodium laurel sulfate, sodiumoctyl glycoside; lauryl-, myristyl-, linoleyl- or stearyl-sulfobetaine:lauryl-, myristyl-, linoleyl- or stearyl-sarcosine; linoleyl-,myristyl-, or cetyl-betaine, lauroamidopropyl-, cocamidopropyl-,linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, orisostearamidopropyl-betaine (e.g. lauroamidopropyl); myristamidopropyl-,palmidopropyl-, or isostearamidopropyl-dimethylamine; sodium methylcocoyl-, or disodium methyl oleyl-taurate; and1-ethyl-1-(2-hydroxyethyl)-2-isoheptadecylimidazolinium ethylsulfate(e.g., the MONAQUAT™ series, Mona Industries, Inc., Paterson, N.J.).Non-limiting examples of commercial products comprising compoundssimilar to TRITON™ X-100 include CONCO™ N1, DOWFAX™ 9N, IGEPAL™ CO,MAKON™, NEUTRONYX® 600's, NONIPOL™ NO, POLYTERGENT® B, RENEX™ 600 's,SOLAR™ NO, STEROX™, SERFONIC™ N, T-DET-N™, TERGITOL™ NP, and TRITON™ N.

The term “antibody” is used to mean an immunoglobulin molecule thatrecognizes and specifically binds to a target, such as a protein,polypeptide, peptide, carbohydrate, polynucleotide, lipid, orcombinations of the foregoing etc., through at least one antigenrecognition site within the variable region of the immunoglobulinmolecule. As used herein, the term encompasses intact polyclonalantibodies, intact monoclonal antibodies, antibody fragments (such asFab, Fab′, F(ab′)2, and Fv fragments), single chain Fv (scFv) mutants,multispecific antibodies such as bispecific antibodies generated from atleast two intact antibodies, monovalent or monospecific antibodies,chimeric antibodies, humanized antibodies, human antibodies, fusionproteins comprising an antigen determination portion of an antibody, andany other modified immunoglobulin molecule comprising an antigenrecognition site so long as the antibodies exhibit the desiredbiological activity. An antibody can be any of the five major classes ofimmunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes)thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), based on theidentity of their heavy-chain constant domains referred to as alpha,delta, epsilon, gamma, and mu, respectively.

The term “Fe-containing polypeptide” as used herein refers to a proteinin which one or more polypeptides are linked to, an Fc region or avariant or derivative thereof. The term “Fc” or “Fc region” refers to aC-terminal region of an IgG heavy chain, including any functionalvariants of IgG Fc that retains the ability of binding to Protein A. Oneexample of an Fe-containing polypeptide is ENBREL® (etanercept) which isa fusion protein fusing a tumor necrosis factor (TNF) receptor to theconstant end of the IgG1 antibody.

The term “CH2/CH3-containing polypeptide” as used herein refers to aprotein in which one or more polypeptides are linked to the CH2/CH3domains of an IgG heavy chain, or a functional variant or derivativethereof.

A “fusion” “chimeric” protein comprises a first amino acid sequencelinked to a second amino acid sequence with which it is not naturallylinked in nature. The amino acid sequences which normally exist inseparate proteins can be brought together in the fusion polypeptide, orthe amino acid sequences which normally exist in the same protein can beplaced in a new arrangement in the fusion polypeptide, e.g., fusion of aFactor VIII domain of the invention with an immunoglobulin Fc domain. Afusion protein is created, for example, by chemical synthesis, or bycreating and translating a polynucleotide in which the peptide regionsare encoded in the desired relationship. A chimeric protein can furthercomprises a second amino acid sequence associated with the first aminoacid sequence by a covalent, non-peptide bond or a non-covalent bond.

The term “linked” as used herein refers to a first amino acid sequencecovalently or non-covalently joined to a second amino acid sequence. Theterm “covalently linked” or “covalent linkage” refers to a covalentbond, e.g., a disulfide bond, a peptide bond, or one or more aminoacids, e.g., a linker, between the two moieties that are linkedtogether. The first amino acid sequence can be directly joined orjuxtaposed to the second amino acid sequence or alternatively anintervening sequence can covalently join the first sequence to thesecond sequence. The term “linked” means not only, a fusion of a firstamino acid sequence to a second amino acid sequence at the C-terminus orthe N-terminus, but also includes insertion of the whole first aminoacid sequence (or the second amino acid sequence) into any twoamino-acids in the second amino acid sequence (or the first amino acidsequence, respectively).

The term “heterologous moiety” refers to a polypeptide or other moietywhich is derived from a distinct entity from that of the entity to whichit is being, compared. For instance, a heterologous polypeptide can besynthetic, or derived from a different species, different cell type ofan individual, or the same or different type of cell of distinctindividuals. In one embodiment, a heterologous moiety can be apolypeptide fused to another polypeptide to produce a fusion polypeptideor protein. In another embodiment, a heterologous moiety can be anon-polypeptide such as PEG conjugated to al polypeptide or protein.

The term “monomer-dimer hybrid” used herein refers to a chimeric proteincomprising a first polypeptide chain and a second polypeptide chain,which are associated with each other by a covalent bond, wherein thefirst chain comprises a biologically active molecule, e.g., a clottingfactor such as Factor IX, Factor VIII, or Factor VII, and an Fc region,and the second chain comprises, consists essentially of, or consists ofan Fc region without the clotting factor. The monomer-dimer hybridconstruct thus is a hybrid comprising a monomer aspect having only onebiologically active molecule and a dimer aspect having two Fe regions.

As used herein, the term “half-life” refers to a biological half-life ofa particular polypeptide in vivo. Half-life can be represented by thetime required for half the quantity administered to a subject to becleared from the circulation and/or other tissues in the animal.

The term “hybridoma” as used herein refers to a cell created by fusionof an immortalized cell derived from an immunologic source and anantibody-producing cell. The resulting hybridoma s an immortalized cellthat produces antibodies. The individual cells used to create thehybridoma can be from any mammalian source, including, but not limitedto, rat, hamster, pig, rabbit, sheep, pig, goat, and human. The termalso encompasses trioma cell lines, which result when progeny ofheterohybrid myeloma fusions, which are the product of a fusion betweenhuman cells and a murine myeloma cell line, are subsequently fused witha plasma cell. Furthermore, the term is meant to include anyimmortalized hybrid cell line that produces antibodies such as, forexample, quadromas (See, e.g., Milstein et al., Nature, 537:3053(1983)).

II. Production and Purification of Polypeptides of Interest A.Polypeptides of Interest

The present invention can be used to inactivate virus that is presentduring production of any polypeptide that is expressed in a host cell.The polypeptide can be expressed from a gene that is endogenous to thehost cell, or from a gene that is introduced into the host cell throughgenetic engineering. The polypeptide can be one that occurs in nature,or can alternatively have a sequence that was engineered or selected bythe hand of man. An engineered polypeptide can be assembled from other,polypeptide segments that individually occur in nature, or can includeone or more segments that are not naturally occurring.

A polypeptide of interest often has a desirable biological or chemicalactivity. For example, the present invention can be employed toinactivate virus that is present during the production of apharmaceutically or commercially relevant enzyme, receptor, antibody,hormone, regulatory factor, antigen, binding agent, etc.

The following is a detailed description of some of the polypeptides thatcan be expressed in a cell culture and purified in accordance with thevirus inactivation method of the present invention.

Clotting Factors

In some embodiments, the protein of interest comprises a clottingfactor. Clotting factor, as used herein, means any molecule, or analogthereof, which prevents or decreases the duration of a bleeding episodein a subject with a hemostatic disorder. For example, a clotting factorused in the invention can be a fall-length clotting factor, a matureclotting factor, or a chimeric clotting factor. In other words, it meansany molecule having clotting activity. Clotting activity, as usedherein, means the ability to participate in a cascade of biochemicalreactions that culminates in the formation of a fibrin clot and/orreduces the severity, duration or frequency of hemorrhage or bleedingepisode. Examples of clotting factors can be found in U.S. Pat. No.7,404,956, which is herein incorporated by reference.

The clotting factor can be a factor that participates in the extrinsicpathway. The clotting factor can be a factor that participates in theintrinsic pathway. Alternatively, the clotting factor can be a factorthat participates in both the extrinsic and intrinsic pathway.

Non-limiting examples of clotting factors include factor I (fibrinogen),factor II (prothrombin), Tissue factor, factor V (proaccelerin, labilefactor), factor VII (stable factor, proconvertin), factor VIII(Antihemophilic factor A), factor IX (Antihemophilic factor B orChristmas factor), factor X (Stuart-Prower factor), factor XI (plasmathromboplastin antecedent), factor XII (Hageman factor), factor XIII(fibrin-stabilizing factor), von Willebrand Factor (VWF), prekallikrein(Fletcher factor), high-molecular-weight kininogen (HMWK) (Fitzgeraldfactor), fibronectin, antithrombin III, heparin cofactor II, protein C,protein S, protein Z, plasminogen, alpha 2-antiplasmin, tissueplasminogen activator (tPA), urokinase, plasminogen activatorinhibitor-1 (PAI1), and plasminogen activator inhibitor-2 (PAI2).

In one embodiment, the clotting factor can be a human clotting factor ora non-human clotting factor, e.g., derived from a non-human primate, apig or any mammal. The clotting factor can be chimeric clotting factor,e.g., the clotting factor can comprise a portion of a human clottingfactor and a portion of a porcine clotting factor or a portion of afirst non-human clotting factor and a portion of a second non-humanclotting factor.

In another embodiment, the clotting factor can be an activated clottingfactor. Alternatively, the clotting factor can be an inactive form of aclotting factor, e.g., a zymogen. The inactive clotting factor canundergo activation subsequent to being linked to at least a portion ofan immunoglobulin constant region. The inactive clotting factor can beactivated subsequent to administration to a subject. Alternatively, theinactive clotting factor can be activated prior to administration.

Factor FIX

“Factor IX protein” or “FIX protein” as used herein, means functionalFactor FIX protein in its normal role in coagulation, unless otherwisespecified. Thus, the FIX polypeptide includes variant polypeptides thatare functional and the polynucleotides that encode such functionalvariant polypeptides. In one embodiment, the FIX polypeptides are thehuman, bovine, porcine, canine, feline, and murine FIX polypeptides. Thefull length polypeptide and polynucleotide sequences of FIX are known,as are many, functional variants, e.g., fragments, mutants and modifiedversions. FIX polypeptides include full-length FIX, full-length FIXminus Met at the N-terminus, full-length FIX minus the signal sequence,mature FIX (minus the signal sequence and propeptide), and mature FIXwith an additional Met at the N-terminus. FIX can be made by recombinantmeans (“recombinant Factor IX” or “rFIX”), i.e., it is not naturallyoccurring or derived from plasma.

A great many functional FIX variants are known. Internationalpublication number WO 02/040544 A3, which is herein incorporated byreference in its entirety, discloses mutants that exhibit increasedresistance to inhibition by heparin at page 4, lines 9-30 and page 15,lines 6-31. International publication number WO 03/020764 A2, which isherein incorporated by reference in its entirety, discloses FIX mutantswith reduced T cell immunogenicity in Tables 2 and 3 (on pages 14-24),and at page 12, lines 1-27. International publication number WO2007/149406 A2, which is herein incorporated by reference in itsentirety, discloses functional mutant FIX molecules that exhibitincreased protein stability, increased in vivo and in vitro half-life,and increased resistance to proteases at page 4, line 1 to page 19, line11. WO 2007/149406 A2 also discloses chimeric and other variant FIXmolecules, at page 19, line 12 to page 20, line 9. Internationalpublication number WO 08/118507 A2, which is herein incorporated byreference in its entirety, discloses FIX mutants that exhibit increasedclotting activity at, page 5, line 14 to page 6, line 5. Internationalpublication number WO 09/051717 A2, which is herein incorporated byreference in its entirety, discloses FIX mutants having an increasednumber of N-linked and/or O-linked glycosylation sites, which results inan increased half-life and/or recovery at page 9, line 11 to page 20,line 2. International publication number WO 09/137254 A2, which isherein incorporated by reference in its entirety, also discloses FactorIX mutants with increased numbers of glycosylation sites at page 2,paragraph [006] to page 5, paragraph [011] and page 16, paragraph [044]to page 24, paragraph [057]. International publication number WO09/130198 A2 which is herein incorporated by reference in its entirety,discloses functional mutant FIX molecules that have an increased numberof glycosylation sites, which result in an increased half-life, at page4, line 26 to page 12, line 6. International publication number WO09/140015 A2, which is herein incorporated by reference in its entirety,discloses functional FIX mutants that an increased number of Cysresidues, which can be used for polymer (e.g., PEG) conjugation, at page11, paragraph [0043] to page 13, paragraph [0053]. The FIX polypeptidesdescribed in International Application No. PCT/US2011/043569 filed Jul.11, 2011 and published as WO 2012/006624 on Jan. 12, 2012 are alsoincorporated herein by reference in its entirety.

In one embodiment, the polypeptide of interest is a long-acting orlong-lasting FIX polypeptide that is a chimeric polypeptide comprising aFIX polypeptide and an FcRn binding partner. In certain embodiments, thepolypeptide of interest is rFIX-Fc which is a fusion protein comprisinga single molecule of human recombinant coagulation FIX (rFIX) covalentlylinked to the dimeric Fc region of immunoglobulin G1 (IgG1) with nointervening sequence. The term “FcRn binding partner” is defined herein.

Factor VIII

“Factor VIII protein” or “FVIII protein” as used herein., meansfunctional Factor VIII protein in its normal role in coagulation, unlessotherwise specified. Thus, the term FVIII includes variant proteins thatare functional. In one embodiment, the FVIII protein is the human,porcine, canine, rat, or murine FVIII protein. A functional FVIIIprotein can be a fusion protein, such as, but not limited to, a fusionprotein comprising a fully or partially B domain-deleted FVIII, at leasta portion of an immunoglobulin constant region, e.g., an Fc domain, orboth. Myriad functional FVIII variants have been constructed and can beused as recombinant FVIII proteins as described herein. See PCTPublication Nos. WO 2011/069164 A2, WO 2012/006623 A2, WO 2012/006635A2, or WO 2012/006633 A2, all of which are incorporated herein byreference in their entirety. FVIII can be a single chain FVIII or a dualchain FVIII.

A great many functional FVIII variants are known. In addition, hundredsof nonfunctional mutations in FVIII have been identified in hemophiliapatients. See, e.g., Cutler et al., Hum. Mutat. 19:274-8 (2002),incorporated herein by reference in its entirety. In addition,comparisons between FVIII from humans and other species have identifiedconserved residues that are likely to be required for function. See,e.g., Cameron et al., Thromb. Haemost. 79:317-22 (1998) and U.S. Pat.No. 6,251,632, incorporated herein by reference in their entirety.

The human FVIII amino acid sequence was deduced from cDNA as shown inU.S. Pat. No. 4,965,199, which is incorporated herein by reference inits entirety. Native mature human FVIII derived from the cDNA sequence(i.e., without the secretory signal peptide but prior to otherpost-translational processing) can be found as SEQ ID NO:1 in WO2013/123457 A1, which is incorporated herein by reference in itsentirety. Partially or fully B domain-deleted FVIII is functional andhas been used in commercial FVIII therapeutics. See, e.g., EP 506757 B2,which is incorporated herein by reference in its entirety.

In one embodiment, the polypeptide of interest is a long-acting orlong-lasting FVIII polypeptide that is a chimeric polypeptide comprisinga FVIII polypeptide and an FcRn binding partner. In certain embodiments,the polypeptide of interest is rFVIII-Fc which is a fusion proteincomprising a single molecule of human recombinant coagulation FVIII(rFVIII) covalently linked to the dimeric Fc region of immunoglobulin G1(IgG1) with no intervening sequence.

Factor VII

“Factor VII protein” or “FVII protein” as used herein, means functionalFactor VII protein in its normal role in coagulation, unless otherwisespecified. It can be a mature form of Factor VII or a variant thereof.Factor VII is a serine protease that is part of the coagulation cascade.FVII includes a Gla domain, two EGF domains (EGF-1 and EGF-2), and aserine protease domain (or peptidase S1 domain) that is highly conservedamong all members of the peptidase S1 family of serine proteases, suchas for example with chymotrypsin. FVII occurs as a single chain zymogen,an activated zymogen-like two-chain polypeptide (e.g., activatable FVII)and a fully activated two-chain form.

As used herein, a “zymogen-like” Protein or polypeptide refers to aprotein that has been activated by proteolytic cleavage, but stillexhibits properties that are associated with a zymogen, such as, forexample, low or no activity, or a conformation that resembles theconformation of the zymogen form of the protein. For example, when it isnot bound to tissue factor, the two-chain activated form of FVII is azymogen-like protein; it retains a conformation similar to the uncleavedFVII zymogen, and, thus, exhibits very low activity. Upon binding totissue factor, the two-chain activated form of FVII undergoesconformational change and acquires its full activity as a coagulationfactor.

Exemplary FVII variants include those with increased specific activity,e.g., mutations that increase the activity of FVII by increasing itsenzymatic activity (Kcat or Km). Such variants have been described inthe art and include, e.g., mutant forms of the molecule as described forexample in Persson et al., Proc. Natl. Acad Sci. USA 98:13583 (2001);Petrovan and Ruf, J. Biol. Chem. 276:6616 (2001); Persson et al., J.Biol. Chem. 276:29195 (2001); Soejima et al., J. Biol. Chem. 276:17229(2001); Soejima et al., J. Biol. Chem. 247:49027 (2002).

In one embodiment, the polypeptide of interest is a long-acting orlong-lasting FVII polypeptide that is a chimeric polypeptide comprisinga FVII polypeptide and an FcRn binding partner. In certain embodiments,the polypeptide of interest is rFVII-Fc which is a fusion proteincomprising a single molecule of human recombinant coagulation FVII(rFIX) covalently linked to the dimeric Fc region of immunoglobulin G1(IgG1) with no intervening sequence.

Chimeric Clotting Factors

In certain embodiments, the polypeptide of interest comprises a chimericclotting factor. In certain embodiments, the chimeric clotting factorcomprises a clotting factor and a CH2/CH3 domain. CH2 and CH3 are twoconstant domains located in the Fc region of an IgG heavy chain. The CH2domain of a human IgG Fc region usually extends from amino acids 231 toamino acid 341 according to the numbering system as described in Kabatet al. 1991, Sequences of Proteins of Immunological Interest, U. S.Department of Public Health, Bethesda; MD, incorporated herein byreference in its entirety. The CH3 domain of a human IgG Fc regionusually extends from amino acids 342 to 447 according to the numberingsystem of Kabat et al., 1991. A CH2/CH3 domain includes any functionalderivative or variants of the CH2 and CH3 domains.

In certain embodiments, the chimeric clotting factor comprises aclotting factor and an Fc region. In one embodiment, the chimericclotting factor is FIX-Fc, FVIII-Fc, or FVII-Fc. Various examples ofFIX-Fc, FVIII-Fc, or FVII-Fc chimeric and hybrid polypeptides aredescribed, for example, in U.S. Pub. Nos. 2013/0202595 A1, 2013/0108629A1, and U.S. Pat. No. 8,329,182, which are incorporated herein byreference in their entirety.

In one embodiment, the polypeptide of interest is a long-acting orlong-lasting clotting factor that is a chimeric polypeptide comprising aclotting factor and an FcRn binding, partner. In certain embodiments,the polypeptide of interest is a fusion protein comprising a singlemolecule of human recombinant clotting factor covalently linked to thedimeric Fc region of immunoglobulin G1 (IgG1) with no interveningsequence.

Heterologous Moieties

In certain embodiments, the polypeptide of interest is a chimericpolypeptide comprising a biologically active molecule and at least oneheterologous moiety. In one embodiment, the biologically active moleculeis a clotting factor. In one embodiment, the heterologous moiety iscapable of extending the half-life of the clotting factor.

In certain embodiments, the heterologous moiety is an IgG or a fragmentthereof, an albumin or a fragment thereof, an albumin binding moiety, aPAS sequence, a homo-amino acid polymer (HAP) sequence, transferrin or afragment thereof, and any combinations thereof, or a non-polypeptidemoiety comprising polyethylene glycol (PEG), polysialic acid,hydroxyethyl starch MES), a derivative thereof, and any combinationsthereof.

In one embodiment the heterologous moiety comprises a first Fe region.In another embodiment, the heterologous moiety comprises a second Feregion.

As used herein, the term “Fc region” is defined as the portion of apolypeptide which corresponds to the Fc region of native immunoglobulin,i.e., as formed by the dimeric association of the respective Fc domainsof its two heavy chains. A native Fc region forms a homodimer withanother Fc region.

In one embodiment, the “Fc region” refers to the portion of a singleimmunoglobulin heavy chain beginning in the hinge region just upstreamof the papain cleavage site (i.e. residue 216 in IgG, taking the firstresidue of heavy chain constant region to be 114) and ending at theC-terminus of the antibody. Accordingly, a complete Fc region comprisesat least a hinge domain, a CH2 domain, and a CH3 domain.

The Fc region of an immunoglobulin constant region, depending on theimmunoglobulin isotype can include the CH2, CH3, and CH4 domains, aswell as the hinge region. Chimeric proteins comprising an Fc region ofan immunoglobulin bestow several desirable properties on a chimericprotein including increased stability, increased serum half-life (seeCapon et al., 1989, Nature 337:525) as well as binding to Fc receptorssuch as the neonatal Fc receptor (FcRn) (U.S. Pat. Nos. 6,086,875,6,485,726, 6,030,613; WO 03/077834; US2003-0235536A1), which areincorporated herein by reference in their entirety.

In another embodiment, the “Fc region” includes an amino acid sequenceof an Fc domain or derived from an Fc domain. In certain embodiments, anFc region comprises at least one of a hinge (e.g., upper, middle, and/orlower hinge region) domain (about amino acids 216-230 of an antibody Fcregion according to EU numbering), a CH2 domain (about amino acids231-340 of an, antibody Fe region, according to EU numbering), a CH3domain (about amino acids 341-438 of an antibody Fc region according toEU numbering), a CH4 domain, or a variant, portion, or fragment thereof.In other embodiments, an Fc region comprises a complete Fc domain (i.e.,a hinge domain, a CH2 domain, and a CH3 domain). In some embodiments, anFc region comprises, consists essentially of, or consists of a hingedomain (or a portion thereof) fused to a CH3 domain (or a portionthereof), a hinge domain (or a portion thereof) fused to a CH2 domain(or a portion thereof), a CH2 domain (or a portion thereof) fused to aCH3 domain (or a portion thereof), a CH2 domain (or a portion thereof)fused to both a hinge domain (or a portion thereof) and a CH3 domain (ora portion thereof). In still other embodiments, an Fc region lacks atleast a portion of a CH2 domain (e.g., all or part of a CH2 domain). Ina particular embodiment, an Fc region comprises or consists of aminoacids corresponding to EU numbers 221 to 447.

The Fc regions of the invention may employ art-recognized Fc variantswhich are known to impart a change (e.g., an enhancement or reduction)in effector function and/or FcR or FcRn binding. Specifically, a bindingmolecule of the invention may include, for example, a change (e.g., asubstitution) at one or more of the amino acid positions disclosed inInternational PCT Publications WO88/07089A1, WO96/14339A1, WO98/05787A1,WO98/23289A1, WO99/51642A1, WO99/58572A1, WO00/09560A2, WO00/32767A1,WO00/42072A2, WO02/44215A2, WO02/060919A2, WO03/074569A2, WO04/016750A2,WO04/029207A2, WO04/035752A2, WO04/063351A2, WO04/074455A2,WO04/099249A2, WO05/040217A2, WO04/044859, WO05/070963A1, WO05/077981A2,WO05/092925A2, WO05/123780A2, WO06/019447A1, WO06/047350A2, andWO06/085967A2; US Patent Publication Nos. US2007/0231329,US2007/0231329, US2007/0237765, US2007/0237766, US2007/0237767,US2007/0243188, US20070248603, US20070286859, US20080057056 or U.S. Pat.Nos. 5,648,260; 5,739,277; 5,834,250; 5,869,046; 6,096,871; 6,121,022;6,194,551; 6,242,195; 6,277,375; 6,528,624; 6,538,124; 6,737,056;6,821,505; 6,998,253; 7,083,784; 7,404,956, and 7,317,091, each of whichis incorporated by reference herein. In one embodiment, the specificchange (e.g., the specific substitution of one or more amino acidsdisclosed in the art) may be made at one or more of the disclosed aminoacid positions. In another embodiment, a different change at one or moreof the disclosed amino acid positions (e.g., the different substitutionof one or more amino acid position disclosed in the art) can be made.

In certain embodiments, a chimeric polypeptide used in accordance withthe invention comprises one or more truncated Fc regions that arenonetheless sufficient to confer Fc receptor (FcR) binding properties tothe Fc region. For example, the portion of an Fc region that binds toFcRn (i.e., the FcRn binding portion) comprises from about amino acids282-438 of IgG1, EU numbering (with the primary contact sites beingamino acids 248, 250-257, 272, 285, 288, 290-291, 308-311, and 314 ofthe CH2 domain and amino acid residues 385-387, 428, and 433-436 of theCH3 domain. Thus, an Fc region of the invention may comprise or consistof an FcRn binding portion. FcRn binding portions may be derived fromheavy chains of any isotype, including IgG1, IgG2, IgG3 and IgG4.

FcRn binding partner (“FcRn BP”) comprises functional neonatal Fcreceptor (FcRn) binding partners, unless otherwise specified. An FcRnbinding partner is any molecule that can be specifically bound by theFcRn receptor with consequent active transport by the FcRn receptor ofthe Ran binding partner. Thus, the term FcRn BP includes any variants ofIgG Fc that are functional. For example, the region of the Fc portion ofIgG that binds to the FcRn receptor has been described based on X-raycrystallography (Burmeister et al. 1994, Nature 372:379, incorporatedherein by reference in its entirety). The major contact area of the Fcwith the FcRn is near the junction of the CH2 and CH3 domains. Fc-FcRncontacts are all within a single Ig heavy chain. FcRn BPs include wholeIgG, the Fe fragment of IgG, and other fragments of IgG that include thecomplete binding, region of FcRn. The major contact sites include aminoacid residues 248, 250-257, 272, 285, 288, 290-291, 308-311, and 314 ofthe CH2 domain and amino acid residues 385-387, 428, and 433-436 of theCH3 domain. References made to amino acid numbering of immunoglobulinsor immunoglobulin fragments, or regions, are all based on Kabat al.1991, Sequences of Proteins of Immunological Interest, U. S. Departmentof Public Health, Bethesda; MD, incorporated herein by reference in itsentirety. The FcRn receptor has been isolated from several mammalianspecies including humans. The sequences of the human FcRn, rat FcRn, andmouse FcRn are known (See, e.g., Story et al. 1994, J. Exp. Med. 180:2377, incorporated herein by reference in its entirety). An FcRn BP cancomprise the CH2 and CH3 domains of an immunoglobulin with or withoutthe hinge region of the immunoglobulin. Exemplary FcRn BP variants areprovided in WO 2004/101740 and WO 2006/074199, incorporated herein byreference in its entirety.

In certain embodiments, the heterologous moiety is an albumin or afragment thereof. Human serum albumin (HSA, or HA), a protein of 609amino acids in its full-length form, is responsible for a significantproportion of the osmotic pressure of serum and also functions as acarrier of endogenous and exogenous ligands. The term “albumin” as usedherein includes full-length albumin or a functional fragment, variant,derivative, or analog thereof. Further examples of albumin or thefragments or variants thereof are disclosed in US Pat. Publ. Nos.2008/0194481A1, 2008/0004206 A1, 2008/0161243 A1, 2008/0261877 A1, or2008/0153751 A1 or PCT Appl. Publ. Nos. 2008/035′413 A2, 2009/058322 A1,or 2007/021494 A2, which are herein incorporated by reference in theirentirety.

In certain embodiments, the heterologous moiety is an albumin bindingmoiety, which, comprises an albumin binding peptide, a bacterial albuminbinding domain, an albumin-binding antibody fragment, or anycombinations thereof. For example, the albumin binding protein can be abacterial albumin binding protein, an antibody or an antibody fragmentincluding domain antibodies (see U.S. Pat. No. 6,696,245). An albuminbinding protein, for example, can be a bacterial albumin binding domain,such as the one of streptococcal protein G (Konig, T and Skerra. A.(1998) J. Immunol. Methods 218, 73-83). Other examples of albuminbinding peptides that can be used as conjugation partner are, forinstance, those having a Cys-Xaa 1-Xaa 2-Xaa 3-Xaa 4-Cys consensussequence, wherein Xaa 1 is Asp, Asn, Ser, Thr, or Trp; Xaa 2 is Asn, GlnHis, Ile, Leu, or Lys; Xaa 3 is Ala, Asp, Phe, Trp, or Tyr; and Xaa 4 isAsp, Gly, Leu, Phe, Ser, or Thr as described in US patent application2003/0069395 or Dennis et al. (Dennis et al. (2002) J. Biol. Chem. 277,35035-35043).

In other embodiments, the heterologous moiety is a PAS sequence. A PASsequence, as used herein, means an amino acid sequence comprising mainlyalanine and serine residues or comprising mainly alanine, serine, andproline residues, the amino acid sequence forming random coilconformation under physiological conditions. Accordingly, the PASsequence is a building block, an amino acid polymer, or a sequencecassette comprising, consisting essentially of, or consisting ofalanine, serine, and proline which can be used as a part of theheterologous moiety in the chimeric protein. Yet, the skilled person isaware that an amino acid polymer also may form random coil conformationwhen residues other than alanine, serine, and proline are added as, aminor constituent in the PAS sequence.

Non-limiting examples of the PAS sequences forming random coilconformation comprise an amino acid sequence selected from the groupconsisting of ASPAAPAPASPAAPAPSAPA (SEQ ID NO:17), AAPASPAPAAPSAPAPAAPS(SEQ ID NO:18), APSSPSPSAPSSPSPASPSS (SEQ ID NO:19), APSSPSPSAPSSPSPASPS(SEQ ID NO:20), SSPSAPSPSSPASPSPSSPA (SEQ ID NO:21),AASPAAPSAPPAAASPAAPSAPPA (SEQ ID NO:22) and ASAAAPAAASAAASAPSAAA (SEQ IDNO:23) or any combinations thereof, Additional examples of PAS sequencesare known from, e.g., US Pat. Publ. No 2010/0292130 A1 and PCT Appl.Publ. No. WO 2008/155134 A1, which are herein incorporated by referencein their entirety.

In yet other embodiments, the heterologous moiety is a glycine-richhomo-amino-acid polymer (HAP). The HAP sequence can comprise arepetitive sequence of glycine, which has at least 50 amino acids inlength. In one embodiment, the HAP sequence is capable of extendinghalf-life of a moiety fused to or linked to the HAP sequence.Non-limiting examples of the HAP sequence includes, but are not limitedto (Gly)_(n), (Gly₄Ser)_(n) or S(Gly₄Ser)_(n), wherein n is 1 to 20, 20to 40, or 40 to 200. See, e.g., Schlapschy M et al., Protein Eng. DesignSelection, 20: 273-284 (2007).

In certain embodiments, the heterologous moiety is transferrin or afragment thereof. Any transferrin may be used to make the chimericproteins used in accordance with the invention. As an example, wild-typehuman Tf (Tf) is a 679 amino acid protein, of approximately 75 KDa (notaccounting for glycosylation), with two main domains, N (about 330 aminoacids) and C (about 340 amino acids), which appear to originate from agene duplication. See GenBank accession numbers NM001063, XM002793,M12530, XM039845, XM 039847 and S95936 (ncbi.nlm.nih.gov/), all of whichare herein incorporated by reference in their entirety. Transferrincomprises two domains, N domain and C domain. N domain comprises twosubdomains, N1 domain and N2 domain, and C domain comprises twosubdomains, C1 domain and C2 domain.

In one embodiment, the transferrin portion of the chimeric proteinincludes a transferrin splice variant. In one example, a transferrinsplice variant can be a splice variant of human transferrin, e.g.,Genbank Accession AAA61140. In another embodiment, the transferrinportion of the chimeric protein includes one or more domains of thetransferrin sequence, e.g., N domain, C domain, N1 domain, N2 domain, C1domain, C2 domain or any combinations thereof.

In other embodiments, the heterologous moiety is a soluble polymer knownin the art, including, but not limited to, polyethylene glycol, ethyleneglycol/propylene glycol copolymers, carboxymethylcellulose, dextran, orpolyvinyl alcohol.

The polymer can be of any molecular weight, and can be branched orunbranched. For polyethylene glycol, in one embodiment, the molecularweight is between about 1 kDa and about 100 kDa for ease in handling andmanufacturing. Other sizes may be used, depending on the desired profile(e.g., the duration of sustained release desired, the effects, if any onbiological activity, the ease in handling, the degree or lack ofantigenicity and other known effects of the polyethylene glycol to aprotein or analog). For example, the polyethylene glycol may have anaverage molecular weight of about 200 to about 100,000 kDa.

In some embodiments, the polyethylene glycol may have a branchedstructure. Branched polyethylene glycols are described, for example, inU.S. Pat. No. 5,643,575; Morpurgo et al., Appl. Biochem. Biotechnol.56:59-72 (1996); Vorobjev et al., Nucleosides Nucleotides 18:2745-2750(1999); and Caliceti et al., Bioconjug. Chem. 10:638-646 (1999), each ofwhich is incorporated herein by reference in its entirety.

In certain embodiments, the heterologous moiety is a hydroxyethyl starch(HES) or a derivative thereof. HES is a derivative of naturallyoccurring amylopectin and is degraded by alpha-amylase in the body. HESis a substituted derivative of the carbohydrate polymer amylopectin,which is present in corn starch at a concentration of up to 95% byweight. HES exhibits advantageous biological properties and is used as ablood volume replacement agent and in hemodilution therapy in theclinics (Sommermeyer et al., Krankenhauspharmazie, 8(8), 271-278 (1987);and Weidler et al., Arzneim.-Forschung/Drug Res., 41, 494-498 (1991)).

HES is mainly characterized by the molecular weight distribution and thedegree of substitution. The degree of substitution, denoted as DS,relates to the molar substitution, is known to the skilled people. SeeSommermeyer et al., Krankenhauspharmazie, 8(8), 271-278 (1987), as citedabove, in particular p. 273. In one embodiment, HES has a mean molecularweight (weight mean) of from 1 to 300 kD, from 2 to 200 kD, from 3 to100 kD, or from 4 to 70 kD. HES can further exhibit a molar degree ofsubstitution of from 0.1 to 3, preferably 0.1 to 2, more preferred, 0.1to 0.9, preferably 0.1 to 0.8, and a ratio between C2:C6 substitution inthe range of from 2 to 20 with respect to the hydroxyethyl groups. Incertain embodiments, the heterologous moiety can be mixtures ofhydroxyethyl starches having different mean molecular weights and/ordifferent degrees of substitution and/or different ratios of C2: C6substitution.

In still other embodiments, the non-polypeptide heterologous moiety is apolymer, e.g., polysialic acids (PSAs) or a derivative thereof.Polysialic acids (PSAs) are naturally occurring branched polymers ofsialic acid produced by certain bacterial strains and in mammals incertain cells. Roth J., et al. (1993) in Polysialic Acid: From Microbesto Man, eds. Roth J., Rutishauser U., Troy F. A. (Birkhäauser Verlag,Basel, Switzerland), pp 335-348. They can Le produced in various degreesof polymerization from n=about 80 or more sialic acid residues down ton=2 by limited acid hydrolysis or by digestion with neuraminidases, orby fractionation of the natural, bacterially derived forms of thepolymer. Various methods of attaching or conjugating polysialic acids toa polypeptide have been described (for example, see U.S. Pat. No.5,846,951; WO-A-0187922, and US 2007/0191597 A1, which are incorporatedherein by reference in their entireties.

More detailed description and sequences of the heterologous moietiesthat can be used in this invention is disclosed, for example, in WO2013/123457 A1 and WO 2013/106787 A1, which are incorporated herein byreference in their entirety.

In certain embodiments, the polypeptide of interest is a monomer-dimerhybrid comprising a clotting factor. In one embodiment, themonomer-dimer hybrid is a chimeric, protein comprising a firstpolypeptide chain and a second polypeptide chain, which are associatedwith each other by a disulfide bond, wherein the first chain comprises aclotting factor, e.g., Factor VIII, and an Fc region and the secondchain comprises, consists essentially of or consists of an Fc regionwithout the clotting, factor. Various examples of monomer-dimer hybridscomprising one or more clotting factors are described in U.S. Pat. No.8,329,182, which is incorporated herein by reference in its entirety.

Antibodies

In some embodiments, the polypeptide of interest comprises an antibodyor an antibody fragment. Antibodies are proteins that have the abilityto specifically bind a particular antigen. Any antibody that can beexpressed in a host cell can be used in accordance with the presentinvention. In one embodiment, t e polypeptide of interest is amonoclonal antibody.

Particular antibodies can be made, for example, by preparing andexpressing synthetic genes that encode the recited amino acid sequencesor by mutating human germline genes to provide a gene that encodes therecited amino acid sequences. Moreover, these antibodies can beproduced, e.g., using one or more of the following methods.

Numerous methods are available for obtaining antibodies, particularlyhuman antibodies. One exemplary method includes screening proteinexpression libraries, e.g., phage or ribosome display libraries. Phagedisplay is described, for example, U.S. Pat. No. 5,223,409; Smith (1985)Science 228:1315-1317; WO 92/18619; WO 91/17271; WO 92/20791; WO92/15679; WO 93/01288; WO 92/01047; WO 92/09690; and WO 90/02809. Thedisplay of Fab's on phage is described, e.g., in U.S. Pat. Nos.5,658,727; 5,667,988; and 5,885,793.

In addition to the use of display libraries, other methods can be usedto obtain an antibody. For example, a protein or a peptide thereof canbe used as an antigen in a non-human animal, e.g., a rodent, e.g., amouse, hamster, or rat.

Humanized antibodies can be generated by replacing sequences of the Fvvariable region that are not directly involved in antigen binding withequivalent sequences from human Fv variable regions. General methods forgenerating humanized antibodies are provided by Morrison, S. L. (1985)Science 229:1202-1207, by Oi et al. (1986) BioTechniques 4:214, and byU.S. Pat. No. 5,585,089; U.S. Pat. No. 5,693,761; U.S. Pat. No.5,693,762; U.S. Pat. No. 5,859,205; and U.S. Pat. No. 6,407,213. Thosemethods include isolating, manipulating, and expressing the nucleic acidsequences that encode all or part of immunoglobulin Fv variable regionsfrom at least one of a heavy or light chain. Sources of such nucleicacid are well known to those skilled in the art and, for example, can beobtained from a hybridoma producing an antibody against a predeterminedtarget, as described above, from germline immunoglobulin genes, or fromsynthetic constructs. The recombinant DNA encoding the humanizedantibody can then be cloned into an appropriate expression vector.

The antibodies can be in the form of full length antibodies, or in theform of fragments of antibodies, e.g., Fab, F(ab′)₂, Fd, dAb, and scFvfragments. Additional forms include a protein that includes a singlevariable domain, e.g., a camel or camelized domain. See, e.g., U.S.2005-0079574 and Davies et al. (1996) Protein Eng. 9(6):531-7.

In certain embodiments, the antibody can be an antigen-binding fragmentof a full length antibody, e.g., a Fab, F(ab′)2, Fv or a single chain Fvfragment. Typically, the antibody is, a full length antibody. Theantibody can be a monoclonal antibody or a mono-specific antibody.

In another embodiment, the antibody can be a human, humanized,CDR-grafted, chimeric, mutated, affinity matured, deimmunized, syntheticor otherwise in vitro-generated antibody, and combinations thereof.

The heavy and light chains of the antibody can be substantiallyfull-length. The protein can include at least one, or two, completeheavy chains, and at least one, or two, complete light chains, or scaninclude an antigen-binding fragment (e.g., a Fab, F(ab′)2, Fv or asingle chain Fv fragment). In yet other embodiments, the antibody has aheavy chain constant region chosen from, e.g., IgG1, IgG2, IgG3, IgG4,IgM, IgA1, IgA2, IgD, and IgE; particularly, chosen from, e.g., IgG1,IgG2, IgG3, and IgG4, more particularly, IgG1 (e.g., human IgG1).Typically, the heavy chain constant region is human or a modified formof a human constant region. In another embodiment, the antibody has alight chain constant region chosen from, e.g., kappa or lambda,particularly, kappa (e.g., human kappa).

The methods of the invention can be used to prepare polypeptidescomprising antibodies, human antibodies, humanized antibodies, chimericantibodies, i.e. antibodies having human constant antibodyimmunoglobulin domains coupled to one or more murine variable antibodyimmunoglobulin domain, and/or non-human antibodies, or fragmentsthereof. Specific examples of antibodies suitable for use in the presentinvention include commercially available antibodies such asmuromonab-CD3 (ORTHOCLONE OKT-3 Ortho Biotech), abciximab (REOPRO®,Lilly), rituximab (RITUXAN®, Biogen IDEC), natalizumab (TYSABRI®, BiogenIDEC), dacliximab (ZENAPAX®, Roche Laboratories), basiliximab(SIMULECT®, Novartis), infliximab (REMICADE®, Centocor), palivizumab(SYNAGIS®, MedImmune), trastuzumab (HERCEPTIN®, Genentech), gemtuzumanozogamicin (MYLoTARG™, Wyeth-Ayerst), alemtuzumab (CAMPATH®, Berlex),and any combinations thereof.

Examples of antibodies or antibody/cytotoxin or antibody/luminophoreconjugates contemplated for use in the invention include those thatrecognize one or more of the following antigens: CD2, CD3, CD4, CD8,CD11a, CD14, CD18, C20, CD22, CD23, CD25, CD33, CD40, CD44, CD52, CD80(B7.1), CD86 (B7.2), CD147, IL-4, IL-5, IL-8, IL-10, IL-2 receptor, IL-4receptor, IL-6 receptor, IL-13 receptor, PDGF-β, VEGF, TGF, TGF-β2,TGF-β1, EGF receptor, VEGF receptor, C5 complement, IgE, tumor antigenCA125, tumor antigen MUC1, PEM antigen, LCG (which is a gene productthat is expressed in association with lung cancer), HER-2, atumor-associated glycoprotein TAG-72, the SK-1 antigen, tumor-associatedepitopes; that are present in elevated levels in the sera of patientswith colon and/or pancreatic c cancer-associated epitopes orpolypeptides expressed on breast, colon, squamous cell, prostate,pancreatic, lung, and/or kidney cancer cells and/or on melanoma, glioma,or neuroblastoma cells, TRAIL receptors 1, 2, 3 and 4, the necrotic coreof a tumor, integrin alpha 4 beta 7, the integrin VLA-4, B2 integrins,TNF-α, the adhesion molecule VAP-1, epithelial cell adhesion molecule(EpCAM), intercellular adhesion molecule-3 (ICAM-3), leukointegrinadhesin, the platelet glycoprotein gp IIb/IIIa, cardiac myosin heavychain, parathyroid hormone, rNAPc2 (which is an inhibitor of factorVIIa-tissue factor), MHC I, carcinoembryonic antigen (CEA),alpha-fetoprotein (AFP), tumor necrosis factor (TNF), CTLA-4 (which is acytotoxic T lymphocyte associated antigen), Fc-γ-1 receptor, HLA-DR 10beta, HLA-DR antigen, L-selectin, IFN-γ, Respiratory Syncitial Virus,human immunodeficiency virus (HIV), hepatitis B virus (HBV),Streptococcus mutans, and Staphylococcus aureus.

The methods of the invention can also be, used for anti-idiotypicantibodies, or substantially similar polypeptides, including but notlimited to anti-idiotypic antibodies against: an antibody targeted tothe tumor antigen gp72; an antibody against the ganglioside GD3; or anantibody against the ganglioside GD2.

Receptors

In some embodiments, the polypeptide of interest comprises a receptor.Receptors are typically trans-membrane glycoproteins that function byrecognizing an extra-cellular signaling ligand. Receptors typically havea protein kinase domain in addition to the ligand recognizing domain,which initiates a signaling pathway by phosphorylating targetintracellular molecules upon binding the ligand, leading todevelopmental or metabolic changes within the cell. The receptor can bemodified so as to remove the transmembrane and/or intracellulardomain(s), in place of which there can optionally be attached anIg-domain.

One large family of receptors is the receptor tyrosine kinases (RTKs),The R family includes receptors that are crucial for a variety offunctions numerous cell types (see, e.g., Yarden and Ulrich, Ann. Rev.Biochem. 57:433-478, 1988; Ullrich and Schlessinger, Cell 61:243-254,1990, incorporated herein by reference). Non-limiting examples of RTKsinclude members of the fibroblast growth factor (FGF) receptor family,members of the epidermal growth factor receptor (EGF) family, plateletderived growth factor (PDGF) receptor, tyrosine kinase withimmunoglobulin and EGF homology domains-1 (TIE-1) and TIE-2 receptors(Sato et al., Nature 376(6535):70-74 (1995), incorporated herein byreference) and c-Met receptor, some of which have been suggested topromote angiogenesis, directly or indirectly (Mustonen and Alitalo, J.Cell Biol. 129:895-898, 1995). Other non-limiting examples of RTK'sinclude fetal liver kinase 1 (FLK-1) (sometimes referred to as kinaseinsert domain-containing receptor (KDR) (Terman et al., Oncogene6:1677-83, 1991) or vascular endothelial cell growth factor receptor 2(VEGFR-2)), fins-like tyrosine kinase-1 (Flt-1) (DeVries et al. Science255; 989-991, 1992; Shibuya et al., Oncogene 5:519-524, 1990), sometimesreferred to as vascular endothelial cell growth factor receptor 1(VEGFR-1), neuropilin-1, endoglin, endosialin, and Ax1.

G-Protein Coupled Receptors

In some embodiments, the polypeptide of interest comprises a G-proteincoupled receptor (GPCR). GPCRs are proteins that have seventransmembrane domains. Upon binding of a ligand to a GPCR, a signal istransduced within the cell which results in a change in a biological orphysiological property of the cell.

GPCRs, along with G-proteins and effectors (intracellular enzymes andchannels which are modulated by G-proteins), are the components of amodular signaling system that connects the state of intracellular secondmessengers to extracellular inputs. These genes and gene-products arepotential causative agents of disease.

The GPCR protein superfamily now contains over 250 types of paralogues,receptors that represent variants generated by gene duplications (orother processes), as opposed to orthologues, the same receptor fromdifferent species. The superfamily can be broken down into fivefamilies: Family I, receptors typified by rhodopsin and thebeta2-adrenergic receptor and currently represented by over 200 uniquemembers; Family II, the recently characterized parathyroidhormone/calcitonin/secretin receptor family; Family III, themetabotropic glutamate receptor family in mammals; Family IV, the cAMPreceptor family, important in the chemotaxis and development of D.discoideum; and Family V, the fungal mating pheromone receptors such asSTE2.

Growth Factors and Other Signaling Molecules

In some embodiments, the polypeptide of interests comprises a growthfactor or a signaling molecule. Growth factors are typicallyglycoproteins that are secreted by cells and bind to and activatereceptors on other cells, initiating a metabolic or developmental changein the receptor cell.

CH2/CH3-Containing Polypeptides

Any polypeptide containing a CH2/CH3 domain is suitable for use inaccordance with the present invention. In one embodiment, theCH2/CH3-containing polypeptide is a soluble form of the TNF receptorfused to an Fc region (TNFR-Fc). A commercially available TNFR-Fc isknown as etanercept (ENBREL®, Immunex Corporation), which is a dimericfusion polypeptide consisting of the extracellular ligand-bindingportion of the human 75 kilodalton (p75) tumor necrosis factor receptor(TNFR) linked to the Fc portion of human IgG1. The Fc component ofetanercept contains the constant heavy 2 (CH2) domain, the constantheavy 3 (CH3) domain and hinge region, but not the constant heavy 1(CH1) domain of human IgG1. It is to be understood that an Fc region cancontain one or all of the domains described above. Etanercept isproduced by recombinant DNA technology in a Chinese hamster ovary (CHO)mammalian cell expression system. It consists of 934 amino acids and hasan apparent molecular weight of approximately 150 kilodaltons(Physicians Desk Reference, 2002, Medical Economics Company Inc.).

Other polypeptides that can be purified in accordance with the inventioninclude recombinant fusion polypeptides comprising at least a portion ofan Fe region of an antibody. A polypeptide fused to an Fc domain (e.g.,a CH2/CH3 domain) and identical to or substantially similar to one ofthe following polypeptides is suitable for use in the present disclosedmethod: a flt3 ligand, a CD40 ligand, erythropoietin, thrombopoeitin,calcitonin, Fas ligand, ligand for receptor activator of NF-kappa B(RANKL), tumor necrosis factor (TNF)-related apoptosis-inducing ligand(TRAIL), thymic stroma-derived lymphopoietin, granulocyte colonystimulating factor, granulocyte-macrophage colony stimulating factor,mast cell growth factor, stem cell growth factor, epidermal growthfactor, RANTES, growth hormone, insulin, insulinotropin, insulin-likegrowth factors, parathyroid hormone, interferons, nerve growth factors,glucagon, interleukins 1 through 18, colony stimulating factors,lymphotoxin-β, tumor necrosis factor (TNF), leukemia inhibitory factor,oncostatin-M, various ligands for cell surface molecules ELK and Hek(such as the ligands for eph-related kinases or LERKS), and anycombinations thereof.

Polypeptides suitable for purification according to the invention alsoinclude recombinant fusion polypeptides comprising CH2/CH3 domains of anantibody plus a receptor for any of the above-mentioned polypeptides orpolypeptides substantially similar to such receptors. These receptorsinclude: both forms of TNFR (referred to as p55 and p75). Interleukin-1receptors (type 1 and 2), Interleukin-4 receptor, Interleukin-15receptor, Interleukin-17 receptor, Interleukin-18 receptor,granulocyte-macrophage colony stimulating factor receptor, granulocytecolony stimulating factor receptor, receptors for oncostatin-M andleukemia inhibitory factor, receptor activator of NF-kappa B (RANK),receptors for TRAIL (TRAIL receptors 1, 2, 3, and 4), and receptors thatcomprise death domains, such as Fas or Apoptosis-Inducing Receptor(AIR), as well as any combinations thereof.

Other polypeptides suitable for use in the present method includedifferentiation antigens (referred to as CD polypeptides) or theirligands or polypeptides substantially similar to either of these, whichare fused to CH2/CH3 domains of an antibody. Such antigens are disclosedin Leukocyte Typing VI (Proceedings of the VIth International Workshopand Conference, Kishimoto, Kikutani et al., eds., Kobe, Japan, 1996).Similar CD polypeptides are disclosed in subsequent workshops andconferences in the above referenced proceedings series. Examples of suchantigens include CD27, CD30, CD39, CD40, and ligands thereto (CD27ligand, CD30 ligand, etc.). Several of the CD antigens are members ofthe TNF receptor family, which also includes 41BB ligand and OX40. Theligands are often members of the TNF family, as are 41BB ligand and OX40ligand. Accordingly, members of the TNF and TNFR families can bepurified according to the present invention.

Enzymatically active polypeptides or their ligands can also be purifiedaccording to the invention. Examples include recombinant fusionpolypeptides comprising CH2/CH3 domains of an, antibody fused to all orpart of one of the following polypeptides or their ligands or apolypeptide substantially similar to one of these:metalloproteinase-disintegrin family members, various kinases,glucocerebrosidase, superoxide dismutase, tissue plasminogen activator,Factor VIII, Factor IX, apolipoprotein E, apolipoprotein A-I, globins,an IL-2 antagonist, alpha-1 antitrypsin, TNF-alpha Converting Enzyme,ligands for any of the above-mentioned enzymes, numerous other enzymesand their ligands, and any combinations thereof.

B. Production of Polypeptides of Interest in a Cell Culture

Cells

A polypeptide of interest is first expressed and produced in a host cellculture. Host cells include, but are not limited to, prokaryotic cells,eukaryotic cells, plant cells, yeast cells, animal cells, insect cells,avian cells, mammalian cells, and human cells.

Non-limiting examples of prokaryotic cells that can be used inaccordance with the present invention include bacterial cells, such asGrain-negative or Gram-positive bacteria, for example, Escherichia coli.

Non-limiting examples of mammalian cells that can be used in accordancewith the present invention include human embryonic kidney line (293 or293 cells subcloned, for growth in suspension culture, Graham et al., J.Gen Virol., 36:59 (1977)); BALB/c mouse myeloma line (NSO/1, ECACC No:85110503); human retinoblasts (PER.C6 (CruCell, Leiden, TheNetherlands)); monkey kidney CV1 line transformed by SV40 (COS-7, ATCCCRL 1651); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamsterovary cells (CHO, Urlaub and Chasm, Proc. Natl. Acad. Sci. USA, 77:4216(1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1 587); human cervical carcinoma cells(HeLa, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL5 1); TRI cells (Mather et al., Annals N.Y. Acad. Sci.,383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line(Hep G2).

Additionally, any number of commercially and non-commercially availablehybridoma cell lines that express polypeptides or proteins can beutilized in accordance with the present invention.

The host cells can also be selected or engineered to modify itsposttranslational modification pathways. For example, the cells can beselected or engineered to modify a protein glycosylation pathway.

Cell Culture Processes for Production of Polypeptide of Interest

Various methods of preparing mammalian cells for production of proteinsor polypeptides by batch aid fed-batch culture are well known, in theart. A nucleic acid sufficient to achieve expression (typically a vectorcontaining the gene encoding the polypeptide or protein of interest andany operably linked genetic control elements) can be introduced into thehost cell line by any number of well-known techniques. Typically, cellsare screened to determine which of the host cells have actually taken upthe vector and express the polypeptide or protein of interest.Traditional methods of detecting a particular polypeptide or protein ofinterest expressed by mammalian cells include but are not limited toimmunohistochemistry, immunoprecipitation, flow cytometry,immunofluorescence microscopy, SDS-PAGE, Western blots, enzyme-linkedimmunosorbentassay (ELISA), high performance liquid, chromatography(HPLC) techniques, biological activity assays and affinitychromatography. One of ordinary skill in the art will be aware of otherappropriate techniques for detecting expressed polypeptides or proteins.If multiple host cells express the polypeptide or protein of interest,some or all of the listed techniques can be used to determine which ofthe cells expresses that polypeptide or protein at the highest levels.

C. Purification of Polypeptide of Interest

Procedures for purification of proteins from cell culture initiallydepend on the site of expression of the protein. Some proteins can becaused to be secreted directly from the cell into the surrounding growthmedia; others are made intracellularly. For the latter proteins, thefirst step of a purification process involves lysis of the cell, whichcan be done by a variety of methods, including mechanical shear, osmoticshock, or enzymatic treatments. Such disruption releases the entirecontents of the cell into the homogenate, and in addition producessubcellular fragments that are difficult to remove due to their smallsize. These are generally removed by differential centrifugation or byfiltration. The same problem arises, although on a smaller scale, withdirectly secreted proteins due to the natural death of cells and releaseof intracellular host cell proteins in the course of the proteinproduction run.

Once a clarified solution containing the protein of interest has beenobtained, its separation from the other proteins produced by the cell aswell as from other impurities is usually attempted using a combinationof different chromatography techniques. These techniques separatemixtures of proteins on the basis of their charge, degree ofhydrophobicity, size, or specific binding affinity.

III. Inactivation of Virus During the Protein Purification Process

One critical concern during the protein purification process is theinactivation or removal of viral contaminations. When a polypeptide ofinterest is recombinantly produced in a cell culture, the recombinantDNA encoding the polypeptide must be transfected into theprotein-producing cells. Viruses can, remain in the culture aftertransfection and contaminate the protein samples. Additionally, cellsused for expressing proteins of interest can encode viral, genomes intheir DNA or otherwise contain endogenous viruses, which is anotherpotential source of contamination to a therapeutic product derived fromcells. A biologically-derived therapeutic, such as a polypeptideproduced in a cell culture, must undergo at least two robust viruspurification steps in order to meet the safety, requirements ofregulatory agencies such as the FDA to ensure no active viruses areadministered to a patient.

Several methods are known in the art to inactivate viruses. For example,arginine can be used for virus inactivation, such as the methoddescribed in U.S. Publication No. 2012/0015424 A1, which is incorporatedherein by reference in its entirety. Each method however has its owndisadvantages, and may not be suitable or optimal for some proteinproducts.

When low pH is used to inactivate viruses, it has the potential toprecipitate proteins, cause aggregation of the product, and/or alter theconformation of certain proteins which can lead to product loss. Inaddition, during the protein purification process, the low pH virusinactivation step is typically performed after the protein of interesthas been eluted from the chromatography column and held in a tank orvessel, especially if the target protein is known to elute from thematrix under low pH conditions, resulting in significant product loss.For example, a CH2/CH3-containing polypeptide such as a monoclonalantibody or FIX-Fc is eluted from the Protein A column at pH valuesbelow 4.5.

The present invention provides a novel method of on-column virusinactivation, comprising washing a polypeptide-bound chromatographymatrix with a low pH and high salt wash solution that effectivelyinactivates viruses and maximizes the recovery of the polypeptide.Carrying out the low pH inactivation step on a polypeptide bound to achromatography mat ix improves stability of the polypeptide because thebound polypeptide tends to remain its natural conformation and is unableto aggregate with, each other. In addition, the presence of high salt inthe wash solution significantly reduces the elution of the polypeptideunder low pH conditions.

The present invention provides a method of inactivating virus that ispresent during production of a polypeptide of interest, comprising: (a)binding the polypeptide to a chromatography matrix, and (b) performing avirus inactivation step by washing the polypeptide-bound chromatographymatrix with a wash solution at a pH of lower than about 4.0. The washsolution used in accordance with the present invention comprises asufficient concentration of salt to substantially reduce the elution ofthe polypeptide during the virus inactivation step. The substantialreduction of the polypeptide elution is likely due to enhancedhydrophobic interactions between the polypeptide and the matrix.

The methods of the present invention are useful for inactivating a widerange of enveloped viruses. Viruses that can be inactivated byembodiments of the present invention include, without limitation,enveloped viruses classified such as, for example, mammalian or avianLeukemia viruses, Herpes viruses, Pox viruses, Hepadnaviruses,Flaviviruses, Togaviruses, Coronaviruses, Hepatitis viruses,Retroviruses, Orthomyxoviruses, Paramyxoviruses, Rhadoviruses,Bunyaviruses, Filoviruses, Retroviruses, Encephalitis, Sindbis,Vesicular Stomatitis Virus, Human Immunodeficiency Virus (HIV),Rhinotracheitis, Epstein Barr virus, Cytomegalo Virus, Influenza Virus,Sendai Virus, Vaccinia Virus, or any combinations thereof.

In certain embodiments, the polypeptide of interest is selected from thegroup consisting of: an antibody, a CH2/CH3-containing polypeptide, aclotting factor, a receptor, and any combinations thereof.

In some embodiments, the polypeptide of interest is an antibody or anantibody fragment. In one embodiment, the antibody is a monoclonalantibody. In another embodiment, the antibody is a chimeric antibody, ahuman antibody, or a humanized antibody.

In some embodiments, the polypeptide of interest comprises a clottingfactor. In certain embodiments, the polypeptide of interest is FIX-Fc,FVIII-Fc, or FVII-Fc. In certain embodiments, the polypeptide is amonomer-dimer hybrid. In certain embodiments, the polypeptide furthercomprises a heterologous moiety.

Many chromatography techniques known in the art can be used in thepresent invention. In some embodiments, the chromatography matrix is anaffinity chromatography matrix. In one embodiment, the affinitychromatography matrix is a Protein A column. In yet another embodiment,the Protein A column is selected from the group consisting ofMABSELECT™, MABSELECT™ SuRe, MABSELECT™ SuRe LX, ESHMUNO® A, AMSPHERE™JWT203, TOYOPEARL® AF-rProtein A-650F, PROSEP®-vA Ultra, PROSEP® UltraPlus, PROSEP®-vA High Capacity, and any combinations thereof.Non-limiting examples of chromatography matrix that can be used toimmobilize the Protein A ligand include dextran based matrix, agarosebased matrix, polystyrene based matrix, hydrophilic polyvinyl ethylbased matrix, rigid polymethacrylate based matrix, porous polymer basedmatrix, controlled pore, glass based matrix, and any combinationsthereof.

In some embodiments, the chromatography matrix is a mixed-modechromatography matrix. In one embodiment, the chromatography matrix is amixed-mode anion exchange chromatography matrix. In one embodiment, themixed-mode chromatography matrix is selected from the group consistingof CAPTO™ Adhere, CAPTO™ MMC, ESHMUNO® HCX, CAPTO™ MMC ImpRes, CAPTO™Blue, NUVIA™ cPrime, BLUE SEPHAROSE® Fast Flow, CAPTO™ Adhere ImpRes,CHT™ Ceramic Hydroxyapatite, CFT™ Ceramic Fluoroapatite, and anycombinations thereof. Non-limiting examples of mixed mode chromatographymatrix include dextran based matrix, agarose based matrix, polystyrenebased matrix, polyvinyl ethyl hydrophilic polymer based matrix,macroporous highly crosslinked polymer based matrix, hydroxyapatite((Ca₅(PO₄)₃OH)₂) based matrix, fluoroapatite ((Ca₅(PO₄)₃F)₂) basedmatrix, and any combinations thereof.

In some embodiments, the polypeptide of interest is first harvestedafter recombinantly produced in cell culture. In certain embodiments,the polypeptide is loaded to the chromatography matrix at a pH fromabout 6.0 to about 8.0. In some embodiments, the pH of the loadingbuffer is about 6.0 to about 7.0 or about 7.0 to about 8.0. In oneembodiment, the pH of the loading buffer is about 6.0, about 6.5, about7.0, about 7.5, or about 8.0.

One or more wash steps can be carried out before the polypeptide iseluted from the chromatography matrix. Same or different wash solutionscan be used in these wash steps.

In certain embodiments, the pH of the wash solution is about 2.5 toabout 3.0, about 3.0 to about 3.5, or about 3.5 to about 4.0. In certainembodiments, the pH of the wash solution is about 2.5, about 2.6, about2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.2, about 3.3,about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, orabout 4.0. In one embodiment, the pH of the wash solution is 3.0. Inanother embodiment, the pH of the wash solution is 3.5.

Non-limiting examples of salts that can be added into the solution or toany buffer used in accordance with the present invention include sodiumsalts, potassium salts, calcium salts, magnesium salts, barium salts,zinc salts, aluminum salts, ammonium salts, chloride salts, fluoridesalts, bromide salts, iodide salts, carbonate salts, nitrate salts,phosphate salts, sulfate salts, acetate salts, and combination thereof.In one embodiment, the salt is sodium chloride (NaCl). In anotherembodiment, the salt is ammonium sulfate.

In certain embodiments, the concentration of the salt in the washsolution is greater than about 0.5 M. In some embodiments, theconcentration of the salt in the wash solution is about 0.5 M to about1.0 M, about 1.0 M to about 1.5 M, about 1.5 M to about 2.0 M, about 2.0M to about 2.5 M, about 2.5 M to about 3.0 M, about 3.0 M to about 3.5M, or about 3.5 M to about 4 M. In some embodiments, the concentrationof the salt in the wash solution is about 0.5 M, about 0.6 M, about 0.7M, about 0.8 M, about 0.9 M, about 1.0 M, about 1.1 M, about 1.2 M,about 1.3 M, about 1.4 M, about 1.5 M, about 1.6 M, about 1.7 M, about1.8 M, about 1.9 M, about 2.0 M, about 2.1 M, about 2.2 M, about 2.3 M,about 2.4 M, about 2.5 M, about 2.6 M, about 2.7 M, about 2.8 M, about2.9 M, about 3.0 M, about 3.1 M, about 3.2 M, about 3.3. M, about 3.4 M,about 3.5 M, about 3.6 M, about 3.7 M, about 3.8 M, about 3.9 M, orabout 4.0 M. In a specific embodiment, the salt concentration is about 2M. In another specific embodiment, the salt concentration is about 3 M.

The wash solution can further comprise one or more other components suchas a polymer, an organic solvent, a detergent, arginine, or an argininederivative.

In certain embodiments, the polymer is a polyethylene glycol (PEG), apolypropylene glycol, or a mixture thereof. In one embodiment, thepolymer is PEG. In a specific embodiment, the polymer is PEG 3350. Insome embodiments, the concentration of the polymer is from about 0.1% toabout 20%. In some embodiments, the concentration of the polymer is fromabout 0.1% to about 15%, from 0.1% to about 10%, from about 0.1% toabout 5%, from about 0.1% to about 2%, from about 1% to about 20%, fromabout 1% to about 15%, from about 1% to about 10%, from about 1% toabout 5%, from about 1% to about 2%, from about 5% to about 20%, fromabout 5% to about 15%, from about 5% to about 10%, from about 10% toabout 20%, from about 10% to about 15%, or from about 15% to about 20%.In some embodiments, the concentration of the polymer is about 0.1%,about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%,about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%,or about 20%.

In certain embodiments, the organic solvent is ethanol, methanol,isopropanol, acetone, ethylene glycol, propylene glycol, hexaethyleneglycol, or a mixture thereof. In some embodiments, the concentration ofthe organic solvent is from about 0.1% to about 20%. In someembodiments, the concentration of the organic solvent is from about 0.1%to, about 15%, from 0.1% to about 10%, from about 0.1% to about 5%, fromabout 0.1% to about 2%, from about 1% to about 20%, from about 1% toabout 15%, from about 1% to about 10%, from about 1% to about 5%, fromabout 1% to about 2%, from about 5% to about 20%, from about 5% to about15%, from about 5% to about 10%, from about 10% to about 20%, from about10% to about 15%, or from about 15% to about 20%. In some embodiments,the concentration of the organic solvent IS about 0.1%, about 0.5%,about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%,about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about20%.

In certain embodiments, the detergent is selected from the groupconsisting of octylphenol ethylene oxide condensate (e.g., TRITON™X-100); 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate(CHAPS);3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate(CHAPSO); lauryldimethyl amine oxide (LDAO); polysorbates (e.g.,polysorbates 20 or 80); poloxamers (e.g., poloxamer 188); sodium dodecylsulfate (SDS); sodium laurel sulfate; sodium octyl glycoside; lauryl-,myristyl-, linoleyl- or stearyl-sulfobetaine; lauryl-, myristyl-,linoleyl- or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine;lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-,myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine(e.g., lauroamidopropyl); myristamidopropyl-, palmidopropyl-, orisostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodiummethyl oleyl-taurate;1-ethyl-1-(2-hydroxyethyl)-2-isoheptadecylimidazolinium ethylsulfate(e.g., the MONAQUAT™ series); and any combinations thereof. Otherexamples of commercial products comprising compounds similar to TRITON™X-100 include, but not limited to, CONCO™ NI, DOWFAX™ 9N, IGEPAL™ CO,MAKON™, NEUTRONYX® 600's, NONIPOL™ NO, POLYTERGENT® B, RENEX™ 600's,SOLAR™ NO, STEROX™, SERFONIC™ N, T-DET-N™, TERGITOL™ NP, and TRITON™ N.

In some embodiments, the concentration of the detergent is from about0.01% to about 8%. In some embodiments, the concentration of thedetergent is from about 0.01% to about 7%, from about 0.01% to about 6%,from about 0.01% to about 5%, from about 0.01% to about 4%, from about0.01% to about 3%, from about 0.01% to about 2%, from about 0.01% toabout 1%, from about 0.01% to about 0.5%, from about 0.01% to about0.1%, from about 0.1% to about 8%, from about 0.1% to about 7%, fromabout 0.1% to about 6%, from about 0.1% to about 5%, from about 0.1% toabout 4%, from about 0.1% to about 3%, from about 0.1% to about 2%, fromabout 0.1% to about 1%, from about 0.1% to about 0.5%, from about 0.5%to about 8%, from about 0.5% to about 7%, from about 0.5% to about 6%,from about 0.5% to about 5%, from about 0.5% to about 4%, from about0.5% to about 3%, from about 0.5% to about 2%, from about 0.5% to about1%, from about 1% to about 8%, from about 1% to about 7%, from about 1%to about 6%, from about 1% to about 5%, from about 1% to about 4%, fromabout 1% to about 3%, or from about 1% to about 2%. In some embodiments,the concentration of the detergent is about 0.01%, about 0.05%, about0.1%, about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%,about 6%, about 7%, or about 8%.

In certain embodiments, more than one on-column virus-inactivation stepis carried out during the purification of the polypeptide. In oneembodiment, identical wash solutions are used in multiplevirus-inactivation steps. In another embodiment, different washsolutions are used in multiple virus-inactivation steps.

In some embodiments, at least one of the wash solutions comprisesarginine, an arginine derivative, or a mixture thereof. In someembodiments, the concentration of arginine is from about 0.1 M to about1 M. In some embodiment, the concentration of arginine is about 0.1 M toabout 0.5 M or about 0.5 M to about 1 M. In some embodiments, theconcentration of arginine is about 0.1 M, about 0.2 M, about 0.3 M,about 0.4 M, about 0.5 M, about 0.6 M, about 0.7 M, about 0.8 M, about0.9 M, or about 1 M.

In some embodiments, at least one of the wash solutions comprisesdetergent. In one embodiment, the detergent is lauryldimethyl amineoxide (LDAO). In another embodiment, the detergent is octylphenolethylene oxide condensate (e.g., TRITON™ X-100). In other embodiments,the detergent comprises a compound very similar to TRITON™ X-100 (e.g.,CONCO™ NI, DOWFAX™ 9N, IGEPAL™ CO, MAKON™, NEUTRONYX® 600's, NONIPOL™NO, POLYTERGENT® B, RENEX™ 600's, SOLAR™ NO, STEROX™, SERFONIC™ N,T-DET-N™, TERGITOL™ NP, and TRITON™ N).

In certain embodiments, elution of the polypeptide during the low pHwash step for virus inactivation is reduced to less than 30%. In certainembodiments, elution of the polypeptide during the low pH wash step isreduced to less than 25%, less than 20%, less than 15%, less than 10%,or less than 5%.

After the wash steps, the polypeptide of interest is eluted from thechromatography matrix with an elution solution. In certain embodiments,the pH of the elution solution is less than 4.5. In one embodiment, thepH of the elution solution is about 3.0. In another embodiment, the pHof the elution solution is about 3.4.

In certain embodiments, at least about 70% of the polypeptide isrecovered in the elution solution. In some embodiments, at least about75%, at least about 80%, at least about 85%, at least about 90%, or atleast about 95% of the polypeptide is recovered in the elution solution.

Additional virus inactivation steps can be performed either prior to, orafter, the on-column virus inactivation method disclosed herein.

The eluted polypeptide of interest can be subjected to additionalpurification steps either prior to, or after, the purification methoddisclosed herein. Standard methods include but not limited to,chromatography (e.g., ion exchange, affinity, size exclusion, andhydroxyapatite chromatography), gel filtration, centrifugation, ordifferential solubility, ethanol precipitation or by any other availabletechnique for the purification of proteins (See, e.g., Scopes, ProteinPurification Principles and Practice 2nd Edition, Springer-Verlag, NewYork, 1987; Higgins, S. J. and Hames, B. D. (eds.), Protein Expression:A Practical Approach, Oxford Univ Press, 1999; and Deutscher, M. P.,Simon, M. I., Abelson, J. N. (eds.), Guide to Protein Purification:Methods in Enzymology (Methods in Enzymology Series, Vol 182), AcademicPress, 1997, all incorporated herein by reference). Protease inhibitorssuch as phenyl methyl sulfonyl fluoride (PMSF), leupeptin, pepstatin oraprotinin can be added at any or all stages in order to reduce oreliminate degradation of the polypeptide or protein during thepurification process. Protease inhibitors are particularly desired whencells must be lysed in order to isolate and purify the expressedpolypeptide or protein. One of ordinary skill in the art will appreciatethat the exact purification technique will vary depending on thecharacter of the polypeptide or protein to be purified, the character ofthe cells from which the polypeptide or protein is expressed, and thecomposition of the medium in which the cells were grown.

The virus inactivation method of present invention can be used to helpenable processes that utilize multiple affinity chromatography columns,like simulated moving bed or tandem chromatography which generatemultiple elution pools. Instead of combining the elution pools to carryout low pH viral inactivation which can result in a long holds at low pHvalues and a greater product loss, or going through the tedious andtime-consuming process of carrying out low pH viral inactivation in theindividual pools, the present invention carries out low pH viralinactivation during the individual tandem or simulated moving bedchromatography runs, thus obviating the need for viral inactivation ofthe elution pools.

The foregoing description is to be understood as being representativeonly and is not intended to be limiting. Alternative methods andmaterials for implementing the invention and also additionalapplications will be apparent to one of skill in the art, and areintended to be included within the accompanying claims.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare explained fully in the literature. See, for example, MolecularCloning A Laboratory Manual, 2nd Ed., Sambrook et al., ed., Cold SpringHarbor Laboratory Press: (1989); Molecular Cloning: A Laboratory Manual,Sambrook et al., ed., Cold Springs Harbor Laboratory, New York (1992),DNA Cloning, D. N. Glover ed., Volumes I and II (1985); OligonucleotideSynthesis, M. J. Gait ed., (1984); Mullis et al. U.S. Pat. No.4,683,195; Nucleic Acid Hybridization, B. D. Hames & S. J. Higgins eds.(1984); Transcription And Translation, B. D. Hames & S. J. Higgins eds.(1984); Culture Of Animal Cells, R. I. Freshney, Alan R. Liss, Inc.,(1987); Immobilized Cells And Enzymes, IRL Press, (1986); B. Perbal, APractical Guide To Molecular Cloning (1984); the treatise, Methods InEnzymology, Academic Press, Inc., N.Y.; Gene Transfer Vectors ForMammalian Cells, J. H. Miller and M. P. Calos eds., Cold Spring HarborLaboratory (1987); Methods In Enzymology, Vols. 154 and 155 (Wu et al.eds.); Immunochemical Methods In Cell And Molecular Biology, Mayer andWalker, eds., Academic Press, London (1987); Handbook Of ExperimentalImmunology, Volumes I-IV, D. M. Weir and C. C. Blackwell, eds., (1986);Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., (1986); and in Ausubel et al, Current Protocols inMolecular Biology, John Wiley and Sons, Baltimore, Md. (1989).

All of the references cited above, as well as all references citedherein, are incorporated herein by reference in their entirety.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Example 1 On-Column Viral Inactivation Using a Wash SolutionContaining 2 M Ammonium Sulfate at pH 3.5

The objective of the experiments shown in Examples 1 to 10 is todemonstrate the feasibility and applicability of an on-column low-pHviral inactivation step using ProA and a target polypeptide. Thepolypeptide was bound to the ProA under standard conditions before ahigh salt at neutral pH was applied to the column. A subsequent wash athigh salt and low pH was then applied to inactivate virus while thepolypeptide remained bound to the adsorbent. A series of washes were,performed before the polypeptide was recovered using an elutionsolution.

The goal of the first experiment was to determine if on-column viralinactivation could be performed with minimal recovery loss using 2 Mammonium sulfate wash at, pH 3.5.

In this experiment, a 0.66 cm diameter MABSELECT™ SuRe column (7.2 mL;21 cm) was first equilibrated (EQ) with 4 column volumes (CVs) of 10 mMsodium phosphate (NaPhosphate), 140 mM NaCl, pH 7.4. 256 ml of filtered(at 0.22 μm) harvested cell culture fluid (HCCF) containingrecombinantly produced FIX-Fc was then loaded onto the column (25.5 mgrFIXFc per mL of resin).

The loading was followed by seven wash steps as indicated in Table 1below. The target polypeptide was subsequently eluted with 25 mMcitrate, 150 mM NaCl, pH 3.4. The flow rate of the chromatography wasconsistent at 300 cm/hr or 1.7 ml/min except that a lower rate (100cm/hr) was used during the low pH wash step (wash 4).

TABLE 1 Individual buffers used in Example 1. Step Buffer Components pHVol. EQ 10 mM NaPhosphate, 140 mM NaCl 7.4 4 CVs Load Filtered HCCF 256ml Wash 1 10 mM NaPhosphate, 140 mM NaCl 7.4 4.5 CVs Wash 2 10 mMNaPhosphate, 900 mM NaCl 7.4 4 CVs Wash 3 100 mM Bis-Tris, 2M Ammonium7.0 4 CVs Sulfate Wash 4 100 mM Bis-Tris, 2M Ammonium 3.5 5 CVs SulfateWash 5 100 mM Bis-Tris, 2M Ammonium 7.0 4 CVs Sulfate Wash 6 10 mMNaPhosphate, 900 mM NaCl 7.4 4 CVs Wash 7 10 mM NaPhosphate, 140 mM NaCl7.4 3 CVs Elution 25 mM Citrate, 150 mM NaCl 3.4 3 CVs Strip 10 mMNaPhosphate, 900 mM NaCl 7.4 5 CVs Regeneration 0.1N NaOH 3 CVs HETP* 10mM NaPhosphate, 140 mM NaCl 7.4 4 CVs Storage 500 mM Acetic Acid, 1%Benzyl 3.2 5 CVs Alcohol *HETP stands for Height of an EquivalentTransfer Plate, which is a solution used to measure column integrity.

FIG. 1 is a chromatogram showing the protein concentration and the pH ineach fraction during the chromatography steps. The polypeptide recoverywas 84% in the eluate, which is surprising given the low pH of wash 4(pH=3.5) because such low pH wash would usually lead to polypeptidedissociation from the column and cause substantial product loss. Theviral removal as measured by PCR was 4.96 log₁₀. The combined removaland inactivation provided by the column washes was higher at >6.39log₁₀, indicating the low pH can provide effective inactivation ofviruses.

Samples were assayed for infectious virus by plaque assay and for viralnucleic acids by Q-PCR assay. The column loading was 25.5 mg rFIXFc permL of resin. Tables 2 and 3 summarize the results of X-MLV viralclearance as measured by infectivity and qPCR, respectively.

The load was spiked with 8.79 log₁₀ X-MLV (PFU) and >6.39 log₁₀retroviral inactivation was calculated. When measured by qPCR, the loadwas spiked with 8.77 log₁₀ X-MLV (GC) and a reduction factor of 4.96log₁₀ was calculated. These results show a robust retroviral removal bythe MABSELECT™ SuRe column at high loadings and additional low pH/highsalt buffer. The infectivity results show total retroviral inactivationafter the MABSELECT™ SuRe resin is exposed to one hour of low pH/highsalt buffer.

TABLE 2 X-MLV Clearance Data by MABSELECT ™ SuRe Step by InfectivityLog₁₀ Volume Adjusted Adjusted Log₁₀ Sample Viral Titer Adjust ViralTiter Titer Reduc- Description (PFU/mL) (mL) (PFU) (PFU) tion StockVirus 4.67E+7 263.0 6.14E+8 8.79 0.00 Control Load 2.33E+6 263.0 6.14E+88.79 0.00 Eluate Run 6 <5.99E+0   41.8 <2.50E+2   <2.40 >6.39 PFU =Plaque Forming Units

TABLE 3 X-MLV Clearance Data by MABSELECT ™ SuRe Step by qPCR ViralTiter Volume Adjusted Log₁₀ Sample by qPCR Adjust Viral Titer AdjustedLog₁₀ Description (GC) (mL) (GC) Titer (GC) Reduction Stock Virus4.53E+7 263.0 5.96E+8 8.77 0.00 Control Load 2.26E+6 263.0 5.94E+8 8.770.00 Eluate Run 6 1.53E+2 41.8 6.39E+3 3.81 4.96 GC = Genome Copies

Therefore, the above results demonstrate that a low pH and high saltwash solution can effectively inactivate viruses during a Protein Achromatography purification process without removing the majority of thetarget polypeptide from the column.

Example 2 On-Column Viral Inactivation Using a Wash Solution Containing1 M Arginine HCl at pH 4.7

The goal of this experiment was to compare the effects of on-columnviral inactivation using a wash solution containing 1 M arginine HCl atpH 4.7.

In this experiment, the Protein A column and the target polypeptide werethe same as those in Example 1. A 7.0 mL (20.5 cm) MabSelect SuRe columnwas first equilibrated, and then 157 ml of HCCF containing Fc-fusionprotein was loaded onto the column. Table 4 below summarizes the buffersolutions used in each step. The target polypeptide was bound to the ProA under standard conditions. A subsequent modified wash 3 containingarginine was then applied to inactivate virus while the polypeptideremained bound to the adsorbent. The flow rate of the chromatography wasconsistent at 300 cm/hr or 1.7 ml/min except that a lower rate (100cm/hr or 0.56 ml/min) was used during the arginine wash step (wash 3).

TABLE 4 Individual buffers used in Example 2 Step Buffer Components pHVol. EQ 10 mM NaPhosphate, 140 mM NaCl 7.4 4 CVs Load Filtered HCCF 157ml Wash 1 10 mM NaPhosphate, 140 mM NaCl 7.4 4.5 CVs Wash 2 10 mMNaPhosphate, 900 mM NaCl 7.4 4 CVs Wash 3 1M Arginine HCl 4.7 5 CVs Wash4 10 mM NaPhosphate, 140 mM NaCl 7.4 3 CVs Elution 25 mM Citrate, 150 mMNaCl 3.4 3 CVs Strip 10 mM NaPhosphate, 900 mM NaCl 7.4 5 CVsRegeneration 0.1N NaOH 3 CVs HETP 10 mM NaPhosphate, 140 mM NaCl 7.4 4CVs Storage 500 mM Acetic Acid, 1% Benzyl 3.2 5 CVs Alcohol

FIG. 2 is a chromatogram showing the protein concentration and the pH ineach fraction during the chromatography steps. The polypeptide recoverywas 83% in the eluate, which is comparable to the recovery percentageusing a pH 3.5 and 2 M ammonium sulfate wash solution as shown inExample 1. The viral removal as measured by PCR was >5.54 log₁₀, and thecombined removal and inactivation provided by the column washes washigher at >6.39 log₁₀. Both numbers are very similar to those in Example1.

These results indicate that wash solutions containing either 1 Marginine at pH 4.7 or pH 3.5 with 2 M ammonium sulfate can providesimilar level of viral inactivation and protein recovery.

Example 3 On-Column Viral Inactivation Using a Wash Solution Containing4×CMC Lauryldimethyl Amine Oxide (LDAO)

The goal of this experiment was to compare the effects of on-columnviral inactivation using a wash solution containing 4× critical micelleconcentration (CMC) lauryldimethyl amine oxide (LDAO).

In this experiment, the Protein A column and the target polypeptide werestill the same as those in Example 1. A 7.0 mL (20.5) MABSELECT™ SuRecolumn was equilibrated and then loaded with 256 ml of HCCF containingFe-fusion protein. Table 5 below summarizes the buffer solutions used ineach step. The target polypeptide was bound to the Pro A under standardconditions. A subsequent modified wash 4 containing 4×CMC LDAO was thenapplied to inactivate virus while the polypeptide remained bound to theadsorbent. The flow rate of the chromatography was consistent at 300cm/hr or 1.7 ml/min except that a lower rate (100 cm/hr or 0.56 ml/min)was used during the detergent wash step (wash 4).

TABLE 5 Individual buffers used in Example 3 Step Buffer Components pHVol. EQ 10 mM NaPhosphate, 140 mM NaCl 7.4 4 CVs Load Filtered HCCF 256ml Wash 1 10 mM NaPhosphate, 140 mM NaCl 7.4 4.5 CVs Wash 2 10 mMNaPhosphate, 900 mM NaCl 7.4 4 CVs Wash 3 10 mM NaPhosphate, 140 mM NaCl3 CVs Wash 4 10 mM NaPhosphate, 140 mM NaCl, 7.4 5 CVs 4x CMC LDAO Wash5 10 mM NaPhosphate, 140 mM NaCl 7.4 3 CVs Elution 25 mM Citrate, 150 mMNaCl 3.4 3 CVs Strip 10 mM NaPhosphate, 900 mM NaCl 7.4 5 CVsRegeneration 0.1N NaOH 3 CVs HETP 10 mM NaPhosphate, 140 mM NaCl 7.4 4CVs Storage 500 mM Acetic Acid, 1% Benzyl 3.2 5 CVs Alcohol

FIG. 3 is a chromatogram showing the protein concentration and the pH ineach fraction during the chromatography steps. The polypeptide recoverywas 85% in the eluate, which is comparable to the recovery percentageusing a pH 3.5 and 2 M ammonium sulfate wash solution. The viral removalas measured by PCR was 5.11 log_(in), and the combined removal andinactivation provided by the column washes was higher at >6.40 log₁₀.Both numbers are similar to those in Example 1.

These results indicate that wash solutions containing either 4×CMC LDAOor pH 3.5 with 2 M ammonium sulfate can provide similar level of viralinactivation.

Example 4 On-Column Viral Inactivation Using a Wash Solution Containing20% PEG and 2 M NaCl at pH 3.0

Various low pH wash solutions were used in Examples 4 to 10 to furtherexplore the feasibility and applicability of an on-column viralinactivation step. Protein A chromatography and a monoclonal antibodywere used in the following experiments. The monoclonal antibody wasbound to the ProA under standard conditions before a high salt atneutral pH was applied to the column. A subsequent wash at high salt andlow pH (about pH 3.0) was then applied to inactivate virus while theantibody remained bound to the adsorbent. A series of washes wereperformed before the antibody was recovered using an elution solution.

The goal of this experiment was to determine if on-column viralinactivation could be performed with minimal recovery loss using a pH3.0, 2 M NaCl, 100 mM glycine wash with 20% PEG.

In this experiment, a 0.6 cm diameter MABSELECT™ SuRe column was firstequilibrated (EQ) with 5 column volumes (CVs) of 75 mM sodium phosphate,100 mM NaCl, pH 7.3. 50 ml of filtered (at 0.22 μm) HCCF containing thepolypeptide of interest was then loaded onto the column to ≤35g/L_(resin). The column was chased with 3 CVs of equilibration buffer.

The loading was followed by 5 CVs wash with 100 mM Bis-Tris, 2 M NaCl,pH 7.0 (wash 1), followed by 5 CVs of low pH wash with 100 mM Glycine,20% PEG 3350, 2M NaCl, pH 3.0 (wash 2). The column was then washed with5 CVs of 100 mM Bis-Tris, 2 M NaCl, pH 7.0 (wash 3), followed by 5 CVswash with 100 mM Bis-Tris, pH 7.0 (wash 4). The target polypeptide wassubsequently eluted with 100 mM glycine, pH 3.0 (elution).

FIG. 4 is a chromatogram showing the protein concentration and the pH ineach fraction during the chromatography steps. Table 6 below summarizesthe condition used in each step. The flow rate of the chromatography wasconsistent at 250 cm/hr or 1.42 ml/min except that a lower rate (50cm/hr) was used during the regeneration step. Table 7 summarizes thepercentage recovery calculated for each chromatography step.

TABLE 6 Individual buffers used in Example 4 Step Buffer Components pHVol. Fraction EQ 75 mM NaPhosphate, 100 mM NaCl 7.3 5 CVs Load FilteredHCCF 50 ml F2 Chase 75 mM NaPhosphate, 100 mM NaCl 7.3 3 CVs F2 Wash 1100 mM Bis-Tris, 2M NaCl 7.0 5 CVs F3 Wash 2 100 mM Glycine, 20% PEG3350, 2M NaCl 3.0 5 CVs F4 Wash 3 100 mM Bis-Tris, 2M NaCl 7.0 5 CVs F5Wash 4 100 mM Bis-Tris 7.0 5 CVs F6 Elution 100 mM Glycine 3.0 3.5 CVsF7 Regeneration 0.3N NaOH 5 CVs F8 Flush 75 mM NaPhosphate, 100 mM NaCl7.3 2 CVs Waste Storage 500 mM NaAcetate, 1% Benzyl Alcohol 3.2 4 CVsWaste

TABLE 7 Percentage Recovery in Each Chromatography Step Using A WashSolution Containing 20% PEG and 2M NaCl at pH 3.0 % Recovery Volume ConcMass (calculated from Step (ml) (mg/ml) (mgs) total mass recovered) Load50 F3 Wash 1 33.15 0.2704 8.96 5.5 F4 Wash 2 33.15 0.9514 31.54 19.5 F5Wash 3 33.15 0.0125 0.41 0.3 F6 Wash 4 + pre 41.44 0.0081 0.00 F7Elution 23.205 5.1370 119.20 73.8 F8 Regen 33.15 0.0435 1.44 0.9 TotalMass Recovered (mgs) 161.6 Recoverable Titer Elution Mass/Load 3.23Volume Column Loading (mg/ml resin) 24.5

As shown, in FIG. 4 and Table 7, only minor product loss (19.5% of thetarget polypeptide) was observed during the low pH wash (F4, wash 2)with 20%) PEG and 2 M NaCl at pH 3.0. The majority of the product (73.8%of the target polypeptide) was recovered in the elution buffer (F7).

Example 5 On-Column Viral Inactivation Using a Wash Solution Containing2% Ethanol and 2 M NaCl at pH 3.0

The goal of this experiment was to determine if on-column viralinactivation could be performed with minimal recovery loss using a pH3.0, 2 M NaCl, 100 mM glycine wash with 2% ethanol.

In this experiment, the Protein A column and the target polypeptide werethe same as those in Example 4. All the buffer solutions used in eachstep were also the same as those listed in Table 6, except that the lowpH wash (wash 2) buffer solution was 100 mM glycine, 2% ethanol, 2 MNaCl, pH 3.0. All the flow rates were the same as in Example 4.

FIG. 5 is a chromatogram showing the protein concentration and the pH ineach fraction during the chromatography steps. Table 8 summarizes thepercentage recovery calculated for each chromatography step.

TABLE 8 Percentage Recovery in Each Chromatography Step Using A WashSolution Containing 2% Ethanol and 2M NaCl at pH 3.0. % Recovery VolumeConc Mass (calculated from total Step (ml) (mg/ml) (mgs) mass recovered)Load 50 F3 Wash 1 33.15 0.4013 13.30 7.6 F4 Wash 2 33.15 0.9584 31.7718.2 F5 Wash 3 33.15 0.1419 4.71 2.7 F6 Wash 4 + pre 41.44 0.0111 0.00F7 Elution 23.205 5.3128 123.28 70.7 F8 Regen 33.15 0.0420 1.39 0.8Total Mass Recovered (mgs) 174.5 Recoverable Titer Elution Mass/Load3.49 Volume Column Loading (mg/ml resin) 26.4

As shown in FIG. 5 and Table 8 only minor product loss (18.2% of thetarget polypeptide) was observed during the low pH wash (F4, wash 2)with 20% PEG and 2 M NaCl at pH 3.0. The majority of the product (70.7%of the target polypeptide) was recovered in the elution buffer (F7).

Example 6 On-Column Viral Inactivation Using a Wash Solution Containing2% Ethanol and 2 M Ammonium Sulfate at pH 3.0

The goal of this experiment was to determine if on-column viralinactivation could be performed with minimal recovery loss using a pH3.0, 2 M ammonium sulfate, 100 mM glycine wash with 2% ethanol.

In this experiment, the Protein A column and the target polypeptide werethe same as those in Example 4. All the buffer solutions used in eachstep were also the same as those listed in Table 6, except that the lowpH wash (wash 2) buffer solution was 100 mM glycine, 2% ethano 1, 2 Mammonium sulfate, pH 3.0, and 2 M ammonium sulfate replaced the 2 M NaClin wash 1 and wash 3 solutions. All the flow rates were the same as inExample 4.

FIG. 6 is a chromatogram showing the protein concentration and the pH ineach fraction during the chromatography steps. Table 9 summarizes thepercentage recovery calculated for each chromatography step.

TABLE 9 Percentage Recovery in Each Chromatography Step Using A WashSolution Containing 2% Ethanol and 2M Ammonium Sulfate at pH 3.0. %Recovery Volume Conc Mass (calculated from Step (ml) (mg/ml) (mgs) totalmass recovered) Load 50 F3 Wash 1 33.15 0.1630 5.40 3.1 F4 Wash 2 33.150.0100 0.33 0.2 F5 Wash 3 33.15 0.0016 0.05 0.0 F6 Wash 4 + pre 41.440.0339 0.00 F7 Elution 23.205 6.9053 160.24 92.3 F8 Regen 33.15 0.22687.52 4.3 Total Mass Recovered (mgs) 173.5 Recoverable Titer ElutionMass/Load 3.47 Volume Column Loading (mg/ml resin) 26.3

As shown in FIG. 6 and Table 9, only insignificant product loss (0.2% ofthe target polypeptide) was observed during the low pH wash (F4, wash 2)with 2% ethanol and 2 M ammonium sulfate at pH 3.0. The majority of theproduct (92.3% of the target polypeptide) was recovered in the elutionbuffer (F7).

Example 7 On-Column Viral Inactivation Using a Wash Solution Containing2% Acetone and 2 M Ammonium Sulfate at pH 3.0

The goal of this experiment was to determine if on-column viralinactivation could be performed with minimal recovery loss using a pH3.0, 2 M ammonium sulfate, 100 mM glycine wash with 2% acetone.

In this experiment, the Protein A column and the target polypeptide werethe same as those in Example 4. All the buffer solutions used in eachstep were also the same as those listed in Table 6, except that the lowpH wash (wash 2) buffer solution was 100 mM glycine, 2% acetone, 2 Mammonium sulfate, pH 3.0, and 2 M ammonium sulfate replaced the 2 M NaClin wash 1 and wash 3 solutions. All the flow rates were the same as inExample 4.

FIG. 7 is a chromatogram showing the protein concentration and the pH ineach fraction during the chromatography steps. Table 10 summarizes thepercentage recovery calculated for each chromatography step.

TABLE 10 Percentage Recovery in Each Chromatography Step Using A WashSolution Containing 2% Acetone and 2M Ammonium Sulfate at pH 3.0. %Recovery Volume Conc Mass (calculated from Step (ml) (mg/ml) (mgs) totalmass recovered) Load 50 F3 Wash 1 33.15 0.1495 4.96 2.8 F4 Wash 2 33.150.0100 0.33 0.2 F5 Wash 3 33.15 0.0016 0.05 0.0 F6 Wash 4 + pre 41.440.0339 0.00 F7 Elution 23.205 7.3562 170.70 96.3 F8 Regen 33.15 0.03891.29 0.7 Total Mass Recovered (mgs) 177.3 Recoverable Titer ElutionMass/Load 3.55 Volume Column Loading (mg/ml resin) 26.9

As shown in FIG. 7 and Table 10, only insignificant product loss (0.2%of the target polypeptide) was observed during the low pH wash (F4, wash2) with 2% acetone and 2 M ammonium sulfate at pH 3.0. The majority ofthe product (more than 96.3% of the target polypeptide) was recovered inthe elution, buffer (F7).

Example 8 On-Column Viral Inactivation Using a Wash Solution Containing2 M Ammonium Sulfate at pH 3.0

The goal of this experiment was to determine if on-column viralinactivation could be performed with minimal recovery loss using a pH3.0, 2 M ammonium sulfate, 100 mM glycine wash with no other modifiers.

In this experiment, the Protein A column and the target polypeptide werestill the same as those in Example 4. A 6.6 mL MABSELECT™ SuRe column(19.4 cm) was first equilibrated and then loaded onto 25.8 g/L resinusing 50 mL of antibody in HCCF. A flow rate of 250 cm/hr was usedexcept during the regeneration step (50 cm/hr). All the buffer solutionsused in each step were also the same as those listed in Table 6, exceptthat the low pH wash (wash 2) buffer solution was 100 mM glycine, 2 Mammonium sulfate, pH 3.0, and 2 M ammonium sulfate replaced the 2 M NaClin wash 1 and wash 3 solutions. All the flow rates were the same as inExample 4. The pH 3.0, 2 M ammonium sulfate, 100 mM glycine wash wasused to keep the antibody bound to the resin at low pH. The low pH, highammonium sulfate wash was bracketed by a neutral, high ammonium sulfatewash buffer (pH 7.0, 2 M ammonium sulfate) to ensure that high levels ofammonium sulfate were present as the pH was lowered to 3.0 and also whenit was subsequently raised to 7.0. Without this, significant productelution occurred before and after the wash step. Excess wash buffer wasused at each step to ensure adequate buffer exchange. FIG. 8 is achromatogram showing the protein concentration and the pH in eachfraction during the chromatography steps. Table 11 summarizes thepercentage recovery calculated for each chromatography step.

TABLE 11 Percentage Recovery in Each Chromatography Step Using A WashSolution Containing 2M Ammonium Sulfate at pH 3.0. % Recovery VolumeConc Mass (calculated from Step (ml) (mg/ml) (mgs) total mass recovered)Load 50 F3 Wash 1 33.15 0.1542 5.11 3.0 F4 Wash 2 33.15 0.0954 3.16 1.9F5 Wash 3 33.15 0.0000 0.00 0.0 F6 Wash 4 + pre 41.44 0.0334 0.00 F7Elution 23.205 6.9315 160.85 94.4 F8 Regen 33.15 0.0362 1.20 0.7 TotalMass Recovered (mgs) 170.3 Recoverable Titer Elution Mass/Load 3.41Volume Column Loading (mg/ml resin) 25.8

As shown in FIG. 8 and Table 11, only very minor product loss (1.9% ofthe target polypeptide) was observed during the low pH wash (F4, wash 2)with 2 M ammonium sulfate at pH 3.0. The majority of the product (94.4%of the target polypeptide) was recovered in the elution buffer (F7). Thepercentage of the peak that was monomeric antibody, 96.0%, was notaltered by the high ammonium sulfate wash.

The high salt, low pH wash buffer did not increase removal of host cellprotein (HCP) on the protein A step. The levels of HCP in the eluatewere typically the same or higher than those achieved using a simpleneutral pH, sodium chloride wash. It is likely that the high levels ofammonium sulfate in the low pH wash enhanced interactions between HCPand antibodies or HCP and protein A resin. It is also possible that theammonium sulfate decreased the solubility of the HCP, reducing theclearance.

An additional experiment at conditions similar to the previous one wasperformed but with sodium chlor de substituted for ammonium sulfate. Theyield loss was higher using a 2 M NaCl, pH 3.0 wash step (17.0%)compared to the 2 M ammonium sulfate pH 3.0 wash step (1.9%). Sinceammonium sulfate is a stronger kosmotrope than sodium chloride, a lowerconcentration is required to prevent antibody elution. It is believedthat using a higher concentration of sodium chloride (3 M) would preventantibody elution and allow on-column viral inactivation.

Example 9 On-Column Viral Inactivation Using a Wash Solution Containing2% TRITON™ X-100 and 2 M NaCl at pH 3.0

The goal of this experiment was to determine if on-column viralinactivation could be performed with minimal recovery loss using a pH3.0, 2 M NaCl, 100 mM glycine wash with 2% TRITON™ X-100.

In this experiment, the Protein A column and the target polypeptide werestill the same as those in Example 4. All the buffer solutions used ineach step were also the same as those listed in Table 6, except that thelow pH wash (wash 2) buffer solution was 100 mM glycine, 2% TRITON™X-100, 2 M NaCl, pH 3.0. All the flow rates were the same as in Example4.

FIG. 9 is a chromatogram showing the protein concentration and the pH ineach fraction during the chromatography steps. Table 12 summarizes thepercentage recovery calculated for each chromatography step.

TABLE 12 Percentage Recovery in Each Chromatography Step Using A WashSolution Containing 2% TRITON ™ X-100 and 2M NaCl at pH 3.0. % RecoveryVolume Conc Mass (calculated from Step (ml) (mg/ml) (mgs) total massrecovered) Load 50 F3 Wash 1 33.15 0.4350 14.42 10.3 F4 Wash 2 33.150.0954 3.16 2.3 F5 Wash 3 33.15 0.0000 0.00 0.0 F6 Wash 4 + pre 41.440.0440 0.00 F7 Elution 23.205 5.2009 120.69 86.2 F8 Regen 33.15 0.05151.71 1.2 Total Mass Recovered (mgs) 140.0 Recoverable Titer ElutionMass/Load 2.80 Volume Column Loading (mg/ml resin) 21.2

As shown in FIG. 9 and Table 12, only very minor product loss (2.3% ofthe target polypeptide) was observed during the low pH wash (F4, wash 2)with 2% TRITON™ X-100 and 2 M NaCl at pH 3.0. The majority of theproduct (86.2% of the target polypeptide) was recovered in the elutionbuffer (F7).

Example 10 On-Column Viral Inactivation Using a Wash Solution Containing2 M NaCl at pH 3.0

The goal of this experiment was to, determine if on-column viralinactivation could be performed with minimal recovery loss using a pH3.0, 2 M NaCl, 100 mM glycine wash with no other modifiers.

In this experiment, the Protein A column and the target polypeptide werestill the same as those in Example 4. All the buffer solutions used ineach step were also the same as those listed in Table 6, except that thelow pH wash (wash 2) buffer solution was 100 mM glycine, 2 M NaCl, pH3.0. All the flow rates were the same as in Example 4.

FIG. 10 is a chromatogram showing the protein concentration and the pHin each fraction during the chromatography steps. Table 13 summarizesthe percentage recovery calculated for each chromatography step.

TABLE 13 Percentage Recovery in Each Chromatography Step Using A WashSolution Containing 2M NaCl at pH 3.0. % Recovery Volume Conc Mass(calculated from Step (ml) (mg/ml) (mgs) total mass recovered) Load 50F3 Wash 1 33.15 0.4540 15.05 8.2 F4 Wash 2 33.15 0.9881 32.75 17.9 F5Wash 3 33.15 0.1212 4.02 2.2 F6 Wash 4 + pre 41.44 0.0099 0.00 F7Elution 23.205 5.6518 131.15 71.6 F8 Regen 33.15 0.0086 0.29 0.2 TotalMass Recovered (mgs) 183.3 Recoverable Titer Elution Mass/Load 3.67Volume Column Loading (mg/ml resin) 27.8

As shown in FIG. 10 and Table 13, only minor product loss (17.9% of thetarget polypeptide) was observed during the low pH wash (F4, wash 2)with 2 M NaCl at pH 3.0. The majority of the product (71.6% of thetarget polypeptide) was recovered in the elution buffer (F7).

Taken together, the results in Examples 1 to 10 demonstrate thaton-column viral inactivation using a low pH and high salt wash solutioncould be effectively performed with minimal recovery loss.

Example 11 On-Column Viral Inactivation in a Mixed-Mode Anion ExchangeChromatography Column Using a Low pH Wash Solution Containing 2 MAmmonium Sulfate

The objective of the following experiment is to demonstrate thepotential feasibility and applicability of an on-column viralinactivation step using a low pH and high salt wash solution with othertypes of chromatography such as a mixed mode anion exchangechromatography.

In this experiment, 6 mL of 0.22 micron filtered product pool containinga monoclonal antibody from an anion exchange purification step at 10mg/ml mab was loaded onto a 1.7 mL column containing CAPTO™ Adhere resinat pH 8.0. A high salt wash buffer at pH 8.0 was applied to the column(wash 1). A subsequent modified wash (wash 2-4) was then applied tomaintain the high salt condition but drop the pH to where is suitablefor viral inactivation, while the antibody remained bound to theabsorbent.

Table 14 below shows the conditions used in each step. The flow rate ofthe chromatography was constant at 100 cm/hr.

TABLE 14 Individual buffers used in Example 11. Step Buffer ComponentspH Vol. EQ 50 mM Tris 8.0 15 CVs Load Filtered and concentrated HCCF 6ml Wash 1 50 mM Tris, 2M Ammonium Sulfate 8.0 15 CVs Wash 2 50 mMAcetate, 2M Ammonium Sulfate 5.0 15 CVs Wash 3 50 mM Citrate, 2MAmmonium Sulfate 4.0 15 CVs Wash 4 100 mM Citrate, 2M Ammonium Sulfate3.5 15 CVs Elution 100 mM Citrate 3.4 15 CVs Regeneration 1N NaOH 15 CVsStorage 100 mL/L Benzyl Alcohol, 30.3 g/L 10 CVs Acidic Acid, 0.64 g/LNaOH, 965 mL RO/DI

FIG. 11 is a chromatogram showing the protein concentration and the pHin each fraction during the chromatography steps.

The wash solutions used in this experiment resulted in minimal removalof target polypeptide (20.6%) at these low pHs (3.5 and 4.0). Theoverall recovery was 77.4% in this experiment.

Similar experiment was performed on a standard (not mixed-mode) anionexchange chromatography column using the same low pH and high salt washsolutions. In contrast, the overall recovery of the polypeptide was only49.8% in this experiment, indicating that a significant amount of thepolypeptide was eluted during the low pH wash. This experiment indicatesthat low pH viral inactivation may not be carried out while product isbound to a standard anion exchange resin by using high salt in thebuffer.

Taken together, these results indicate that on-column viral inactivationusing a low pH and high salt wash solution could be effectivelyperformed with minimal recovery loss in certain chromatography methods,such as affinity chromatography and mix-mode anion-exchangechromatography.

Example 12 Inactivation of Viruses

Low pH can damage antibodies. An Fc-fusion protein was inactivated bythe times and pH values required for viral inactivation. A novel,on-column low pH viral inactivation method was developed. The mAbhumanized IgG1 was produced in recombinant Chinese hamster ovary (CHO)cells grown in serum free medium. As described in this example,antibodies were retained on protein A and CAPTO ADHERE® at pH 3.0 using2 M ammonium sulfate to increase hydrophobic interactions. On-columnxMuLV inactivation was demonstrated on, protein A with 2 M ammoniumsulfate pH 3-3.5, 1 M Arginine, and the detergent LDAO. On-columninactivation helps minimize low pH exposure, eliminates addedconductivity during pool acidification, and automates low pHinactivation steps. On-column inactivation provides benefit forsemi-continuous multi-column chromatography which generates many low pHinactivation pools. As described in this example antibodies wereretained on CAPTO MMC® at pH 8.0 using 2 M ammonium sulfate to increasehydrophobic interactions.

The Fc-fusion protein was produced in HEK293 cells grown in serum freemedium. Xenotropic murine leukemia virus (X-MLV) was measured with PCRand infectivity based assays. Table 15 shows xMulV virus inactivationand removal provided by protein A wash buffers with an Fc-fusionprotein.

TABLE 15 xMulV virus inactivation and removal provided by protein A washbuffers with an Fc-fusion protein. Combined Removal and Inactivation byProtein A wash buffer Virus Removal by PCR Infectivity Low pH 4.96 >6.39(pH 3.5, 2M ammonium sulfate) Arginine >5.54 >6.39 (1M arginine pH 4.8)Detergent 5.11 >6.39 (LDAO 4X CMC)

The protein A ligand binds the CH2 and CH3 domains of an antibody Fcthrough hydrophobic, ionic and hydrogen bond interactions. In one partof this example, a 6.6 mL MABSELECT™ SuRe column (19.4 cm) was loadedwith 50 mL of HCCF with 2.2 mg/ml antibody. The column was washed with100 mM BisTris buffer with 2000 mM ammonium sulfate at pH 6.6. A low pHwash buffer with ammonium sulfate was then, applied (100 mM sodiumcitrate, 2000 mM ammonium sulfate at pH 3.0). The ammonium sulfateconcentration was then reduced to zero over a 9 CV gradient. The flowrate for all steps was 250 cm/hr.

As shown in FIG. 12, the antibody began to desorb at about 1700 mM andwas fully eluted at about 200 mM ammonium sulfate. FIG. 12 shows that atleast 1700 mM ammonium sulfate is required to keep the antibody bound toprotein A at pH 3.0. Since at least 1700 mM ammonium sulfate wasrequired to keep the antibody bound to the resin at low pH, thesolubility of the antibody was measured as a function of pH, antibodyand ammonium sulfate concentration. The antibody was found to be solublein 1000 mM ammonium sulfate but not 1500 mM ammonium sulfate. Theprecipitation was observed with 1500 mM ammonium sulfate at pH valuesbetween 3.5 and 8.0 and with antibody concentrations between 10 and 30mg/mL.

In one part of this example, a 6.6 mL MabSelect SuRe column (19.4 cm)was first equilibrated and then loaded to 25.8 g/L resin using 50 mL ofantibody in HCCF. A flow rate of 250 cm/hr was used except during theregeneration step (50 cm/hr).

A pH 3.0, 2 M ammonium sulfate, 100 mM glycine wash was used to keep theantibody bound to the resin at low pH. The low pH, high ammonium sulfatewash was bracketed by a neutral, high ammonium sulfate wash buffer (pH7.0, 2 M ammonium sulfate) to ensure that high levels of ammoniumsulfate were present as the pH was lowered to 3.0 and also when it was,subsequently raised to 7.0. Without this, significant product elutionoccurred before and after the wash step. Excess wash buffer was used ateach step to ensure adequate buffer exchange. The step sequence andbuffers used are identical to those used in Example 8. The proteinconcentration, conductivity, and pH in each step are shown in FIG. 8.FIG. 13 indicates that 2 M ammonium sulfate wash keeps antibody bound toprotein A at pH 3.0.

In one part of this example, a 1.7 mL (5.0 cm) CAPTO ADHERE® column wasfirst equilibrated and then loaded with 6 mL of 11.4 g/L antibody in pH8.0 50 mM Tris buffer. Subsequently a high salt pH 8.0 wash buffer andthen a high salt pH 3.5 wash buffer were applied to the resin. Excesswash buffer was used at each step to ensure adequate buffer exchange.All steps were performed at 100 cm/hr. See Table 16.

TABLE 16 Step sequence and buffers used to produce FIG. 14 of Example12. Volume (CV Step Buffer pH or ml) Equilibration 50 mM Tris 8.0 15Load 50 mM Tris 8.0 6 mL Wash 1 50 mM Tris, 2M Ammonium Sulfate 8.0 15Wash 2 100 mM Citrate 2M Ammonium Sulfate 3.5 15 Wash 3 50 mM Tris, 2MAmmonium Sulfate 8.0 15 Wash 4 50 mM Tris 8.0 15 Elution 100 mM Citrate3.4 15 Regeneration 1.0N NaOH 15

FIG. 14 shows that 2 M ammonium sulfate wash keeps antibody bound toCAPTO ADHERE® at pH 3.5. As shown in FIG. 14, minimal product loss wasobserved during the 2 M ammonium sulfate, pH 3.5 wash, which issurprising because at this pH both the product and the resin have apositive charge. It is likely that the product remains bound to theresin due to enhanced hydrophobic interactions in this high salt buffer.The majority of the product (79.0%) was recovered in the elution pool.The percentage of the peak that was monomeric antibody (98.4%) was notaltered by the high ammonium sulfate wash.

In one part of this example, a 1.7 mL (5.1 cm) column with CAPTO MMC®mixed mode resin was loaded with to mL of 12.4 mg/mL antibody in a pH4.5, 50 mM citrate buffer. Subsequently a high salt pH 4.5 wash and thena high salt pH 8.0 wash were applied. Excess wash buffer was used ateach step to ensure adequate buffer exchange. The protein concentration,conductivity, and pH in each step are shown in FIG. 15. The buffers usedin each step are shown in Table 17. All steps were performed at 100cm/hr.

TABLE 17 Step sequence and buffers used to produce FIG. 15 of Example12. Volume Step Buffer pH (CV or ml) Equilibration 50 mM Citrate 4.5 15Load 50 mM Citrate 4.5 6 mL Wash 1 50 mM Citrate, 2M Ammonium Sulfate4.5 15 Wash 2 50 mM Tris, 2M Ammonium Sulfate 8.0 15 Elution 50 mM Tris8.0 15 Regeneration 0.1N NaOH 15

FIG. 15 shows that 2 M ammonium sulfate wash keeps antibody bound toCAPTO MMC® at pH 8.0. As shown in FIG. 15, minimal product loss wasobserved during the 2 M ammonium sulfate, pH 8.0 wash, which issurprising because at this pH both the product and the resin have anegative charge and antibody elution would be expected to occur. Thepercent recovery in the eluate pool was 94.1%. It is likely that theproduct remains bound to the resin due to enhanced hydrophobicinteractions in this high salt buffer.

A similar experiment was performed using TMAE HiCap resin. TMAE HiCapresin is an anion exchange resin that lacks the hydrophobic interactionproperties of the CAPTO ADHERE™ resin. Significant product loss occurredduring the low pH washes and the overall recovery was 49.8%. The productthat eluted during the low pH wash precipitated in the column and in theinstrumentation. The TMAE HiCap resin experiment indicates thatkosmotropic salts were not capable of preserving adsorption of the mAbto the anion exchange resin at low pH.

The on-column low pH viral inactivation method shows that antibodieswere retained on protein A and CAPTO ADHERE® at pH 3.0 using 2 Mammonium sulfate to increase hydrophobic interactions. On-column xMuLVinactivation was demonstrated on protein A with 2 M ammonium sulfate pH3-3.5, 1 M Arginine, and the detergent LDAO.

These data show that on-column inactivation helps minimize low pHexposure, eliminates added conductivity during pool acidification,automates low pH inactivation step, and helps enable semi-continuousmulti-column chromatography.

The present invention is not to be limited in scope by the specificembodiments described which are intended as single illustrations ofindividual aspects of the invention, and any compositions or methodswhich are functionally equivalent are within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description and accompanying drawings.Such modifications are intended to fall within the scope of the appendedclaims.

All documents, articles, publications, patents, and patent applicationsmentioned in this specification are herein incorporated by reference tothe same extent as if each individual publication or patent applicationwas specifically and individually indicated to be incorporated byreference.

The present application claims priority to U.S. Provisional ApplicationNo. 61/882,488, filed Sep. 25, 2014 and U.S. Provisional Application No.62/028,657, filed Jul. 24, 2014, which are incorporated herein byreference in their entireties.

TABLE 15 Polynucleotide Sequences of FVIII A. B-Domain Deleted FVIII-Fc(i) B-Domain Deleted FVIII-Fc Chain DNA Sequence (FVIIIsignal peptide underlined, Fc region in bold) (SEQ ID NO: 1,which encodes SEQ ID NO: 2)

 101 actatatgca aagtgatctc ggtgagctgc ctgtggacgc aagatttcct 151 cctagagtgc caaaatcttt tccattcaac acctcagtcg tgtacaaaaa 201 gactctgttt gtagaattca cggatcacct tttcaacatc gctaagccaa 251 ggccaccctg gatgggtctg ctaggtccta ccatccaggc tgaggtttat 301 gatacagtgg tcattacact taagaacatg gcttcccatc ctgtcagtct 351 tcatgctgtt ggtgtatcct actggaaagc ttctgaggga gctgaatatg 401 atgatcagac cagtcaaagg gagaaagaag atgataaagt cttccctggt 451 ggaagccata catatgtctg gcaggtcctg aaagagaatg gtccaatggc 501 ctctgaccca ctgtgcctta cctactcata tctttctcat gtggacctgg 551 taaaagactt gaattcaggc ctcattggag ccctactagt atgtagagaa 601 gggagtctgg ccaaggaaaa gacacagacc ttgcacaaat ttatactact 651 ttttgctgta tttgatgaag ggaaaagttg gcactcagaa acaaagaact 701 ccttgatgca ggatagggat gctgcatctg ctcgggcctg gcctaaaatg 751 cacacagtca atggttatgt aaacaggtct ctgccaggtc tgattggatg 801 ccacaggaaa tcagtctatt ggcatgtgat tggaatgggc accactcctg 851 aagtgcactc aatattcctc gaaggtcaca catttcttgt gaggaaccat 901 cgccaggcgt ccttggaaat ctcgccaata actttcctta ctgctcaaac 951 actcttgatg gaccttggac agtttctact gttttgtcat atctcttccc1001 accaacatga tggcatggaa gcttatgtca aagtagacag ctgtccagag1051 gaaccccaac tacgaatgaa aaataatgaa gaagcggaag actatgatga1101 tgatcttact gattctgaaa tggatgtggt caggtttgat gatgacaact1151 ctccttcctt tatccaaatt cgctcagttg ccaagaagca tcctaaaact1201 tgggtacatt acattgctgc tgaagaggag gactgggact atgctccctt1251 agtcctcgcc cccgatgaca gaagttataa aagtcaatat ttgaacaatg1301 gccctcagcg gattggtagg aagtacaaaa aagtccgatt tatggcatac1351 acagatgaaa cctttaagac tcgtgaagct attcagcatg aatcaggaat1401 cttgggacct ttactttatg gggaagttgg agacacactg ttgattatat1451 ttaagaatca agcaagcaga ccatataaca tctaccctca cggaatcact1501 gatgtccgtc ctttgtattc aaggagatta ccaaaaggtg taaaacattt1551 gaaggatttt ccaattctgc caggagaaat attcaaatat aaatggacag1601 tgactgtaga agatgggcca actaaatcag atcctcggtg cctgacccgc1651 tattactcta gtttcgttaa tatggagaga gatctagctt caggactcat1701 tggccctctc ctcatctgct acaaagaatc tgtagatcaa agaggaaacc1751 agataatgtc agacaagagg aatgtcatcc tgttttctgt atttgatgag1801 aaccgaagct ggtacctcac agagaatata caacgctttc tccccaatcc1851 agctggagtg cagcttgagg atccagagtt ccaagcctcc aacatcatgc1901 acagcatcaa tggctatgtt tttgatagtt tgcagttgtc agtttgtttg1951 catgaggtgg catactggta cattctaagc attggagcac agactgactt2001 cctttctgtc ttcttctctg gatatacctt caaacacaaa atggtctatg2051 aagacacact caccctattc ccattctcag gagaaactgt cttcatgtcg2101 atggaaaacc caggtctatg gattctgggg tgccacaact cagactttcg2151 gaacagaggc atgaccgcct tactgaaggt ttctagttgt gacaagaaca2201 ctggtgatta ttacgaggac agttatgaag atatttcagc atacttgctg2251 agtaaaaaca atgccattga accaagaagc ttctctcaaa acccaccagt2301 cttgaaacgc catcaacggg aaataactcg tactactctt cagtcagatc2351 aagaggaaat tgactatgat gataccatat cagttgaaat gaagaaggaa2401 gattttgaca tttatgatga ggatgaaaat cagagccccc gcagctttca2451 aaagaaaaca cgacactatt ttattgctgc agtggagagg ctctgggatt2501 atgggatgag tagctcccca catgttctaa gaaacagggc tcagagtggc2551 agtgtccctc agttcaagaa agttgttttc caggaactta ctgatggctc2601 ctttactcag cccttatacc gtggagaact aaatgaacat ttgggactcc2651 tggggccata tataagagca gaagttgaag ataatatcat ggtaactttc2701 agaaatcagg cctctcgtcc ctatuccttc tattctagcc ttatttctta2751 tgaggaagat cagaggcaag gagcagaacc Lagaaaaaac tttgtcaagc2801 ctaatgasac caaaacttac ttttggaaag tgcaacatca tatggcaccc2851 actaaagatg agtttgactg caaagcctgg gcttatttct ctgatgttga2901 cctggaaaaa gatgtgcact caggcctgat tggacccctt ctggtctgcc2951 acactaacac actgaaccct gctcatggga gacaagtgac agtacaggaa3001 tttgctctgt ttttcaccat ctttgatgag accaaaagct ggtacttcac3051 tgaaaatatg gaaagaaact gcagggctcc ctgcaatatc cagatggaag3101 atcccacttt taaagagaat tatcgcttcc atgcaatcaa tggctacata3151 atggatacac tacctggctt agtaatggct caggatcaaa ggattcgatg3201 gtatctgctc agcatgggca gcaatgaaaa catccattct attcatttca3251 gtggacatgt gttcactgta cgaaaaaaag aggagtataa aatggcactg3301 tacaatctct atccaggcgt ttttgagaca gtggaaatgt taccatccaa3351 agctggaatt tggcgggtgg aatgccttat tggcgagcat ctacatgctg3401 ggatgagcac actttttctg gtgtacagca ataagtgtca gactcccctg3451 ggaatggctt ctggacacat tagagatttt cagattacag cttcaggaca3501 atatggacag tgggccccaa agctggccag acttcattat tccggatcaa3551 tcaatgcctg gagcaccaag gagccctttt cttggatcaa ggtggatctg3601 ttggcaccaa tgattattca cggcatcaag acccagggtg cccgtcagaa3651 gttctccagc ctctacatct ctcagtttat catcatgtat agtcttgatg3701 ggaagaagtg gcagacttat cgaggaaatt ccactggaac cttaatggtc3751 ttctttggca atgtggattc atctgggata aaacacaata tttttaaccc3801 tccaattatt gctcgataca tccgtttgca cccaactcat tatagcattc3851 gcagcactct tcgcatggag ttgatgggct gtgatttaaa tagttgcagc3901 atgccattgg gaatggagag taaagcaata tcagatgcac agattactgc3951 ttcatcctac tttaccaata tgtttgccac ctggtctcct tcaaaagctc4001 gacttcacct ccaagggagg agtaatgcct ggagacctca ggtgaataat4051 ccaaaagagt ggctgcaagt ggacttccag aagacaatga aagtcacagg4101 agtaactact cagggagtaa aatctctgct taccagcatg tatgtgaagg4151 agttcctcat ctccagcagt caagatggcc atcagtggac tctctttttt4201 cagaatggca aagtaaaggt ttttcaggga aatcaagact ccttcacacc4251 tgtggtgaac tctctagacc caccgttact gactcgctac cttcgaattc4301 acccccagag ttgggtgcac cagattgccc tgaggatgga ggttctgggc4351 tgcgaggcac aggacctcta cgacaaaact cacacatgcc caccgtgccc4401 agctccagaa ctcctgggcg gaccgtcagt cttcctcttc cccccaaaac4451 ccaaggacac cctcatgatc tcccggaccc ctgaggtcac atgcgtggtg4501 gtggacgtga gccacgaaga ccctgaggtc aagttcaact ggtacgtgga4551 cggcgtggag gtgcataatg ccaagacaaa gccgcgggag gagcagtaca4601 acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg4651 ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc4701 ccccatcgag aaaaccatct ccaaagccaa agggcagccc cgagaaccac4751 aggtgtacac cctgccccca tcccgggatg agctgaccaa gaaccaggtc4801 agcctgacct gcctggtcaa aggcttctat cccagcgaca tcgccgtgga4851 gtgggagagc aatgggcagc cggagaacaa ctacaagacc acgcctcccg4901 tgttggactc cgacggctcc ttcttcctct acagcaagct caccgtggac4951 aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga5001 ggatctgcac aaccactaca cgcagaagag cctctccctg tctccgggta 5051 aa(ii) Fc DNA sequence mouse Igκ signal peptide underlined)(SEQ ID NO: 3, which encodes SEQ ID NO: 4)

 101 tcctgggagg accgtcagtc ttcctcttcc ccccaaaacc caaggacacc 151 ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg tggacgtgag 201 ccacgaagac cctgaggtca agttcaactg gtacgtggac ggcgtggagg 251 tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 301 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa 351 ggagtacaag tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga 401 aaaccatctc caaagccaaa gggcagcccc gagaaccaca ggtgtacacc 451 ctgcccccat cccgcgatga gctgaccaag aaccaggtca gcctgacctg 501 cctggtcaaa ggcttctatc ccagcgacat cgccgtggag tgggagagca 551 atgggcagcc ggagaacaac tacaagacca cgcctcccgt gttggactcc 601 gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg 651 gcagcagggg aacgtcttct catgctccgt gatgcatgag gctctgcaca 701 accactacac gcagaagagc ctctccctgt ctccgggtaa aB. Full Length FVIII-Fc(i) Full Length FVIII-Fc DNA Sequence (FVIII signal peptideunderlined, Fc region in bold) (SEQ ID NO: 5, which encodesSEQ ID NO: 6)

 101 actatatgca aagtgatctc ggtgagctgc ctgtggacgc aagatttcct 151 cctagagtgc caaaatcttt tccattcaac acctcagtcg tgtacaaaaa 201 gactctgttt gtagaattca cggatcacct tttcaacatc gctaagccaa 251 ggccaccctg gatgggtctg ctaggtccta ccatccaggc tgaggtttat 301 gatacagtgg tcattacact taagaacatg gcttcccatc ctgtcagtct 351 tcatgctgtt ggtgtatcct actggaaagc ttctgaggga gctgaatatg 401 atgatcagac cagtcaaagg gagaaagaag atgataaagt cttccctggt 451 ggaagccata catatgtctg gcaggtcctg aaagagaatg gtccaatggc 501 ctctgaccca ctgtgcctta cctactcata tctttctcat gtggacctgg 551 taaaagactt gaattcaggc ctcattggag ccctactagt atgtagagaa 601 gggagtctgg ccaaggaaaa gacacagacc ttgcacaaat ttatactact 651 ttttgctgta tttgatgaag ggaaaagttg gcactcagaa acaaagaact 701 ccttgatgca ggatagggat gctgcatctg ctcgggcctg gcctaaaatg 751 cacacagtca atggttatgt aaacaggtct ctgccaggtc tgattggatg 801 ccacaggaaa tcagtctatt ggcatgtgat tggaatgggc accactcctg 851 aagtgcactc aatattcctc gaaggtcaca catttcttgt gaggaaccat 901 cgccaggcgt ccttggaaat ctcgccaata actttcctta ctgctcaaac 951 actcttgatg gaccttggac agtttctact gttttgtcat atctcttccc1001 accaacatga tggcatggaa gcttatgtca aagtagacag ctgtccagag1051 gaaccccaac tacgaatgaa aaataatgaa gaagcggaag actatgatga1101 tgatcttact gattctgaaa tggatgtggt caggtttgat gatgacaact1151 ctccttcctt tatccaaatt cgctcagttg ccaagaagca tcctaaaact1201 tgggtacatt acattgctgc tgaagaggag gactgggact atgctccctt1251 agtcctcgcc cccgatgaca gaagttataa aagtcaatat ttgaacaatg1301 gccctcagcg gattggtagg aagtacaaaa aagtccgatt tatggcatac1351 acagatgaaa cctttaagac tcgtgaagct attcagcatg aatcaggaat1401 cttgggacct ttactttatg gggaagttgg agacacactg ttgattatat1451 ttaagaatca agcaagcaga ccatataaca tctaccctca cggaatcact1501 gatgtccgtc ctttgtattc aaggagatta ccaaaaggtg taaaacattt1551 gaaggatttt ccaattctgc caggagaaat attcaaatat aaatggacag1601 tgactgtaga agatgggcca actaaatcag atcctcggtg cctgacccgc1651 tattactcta gtttcgttaa tatggagaga gatctagctt caggactcat1701 tggccctctc ctcatctgct acaaagaatc tgtagatcaa agaggaaacc1751 agataatgtc agacaagagg aatgtcatcc tgttttctgt atttgatgag1801 aaccgaagct ggtacctcac agagaatata caacgctttc tccccaatcc1851 agctggagtg cagcttgagg atccagagtt ccaagcctcc aacatcatgc1901 acagcatcaa tggctatgtt tttgatagtt tgcagttgtc agtttgtttg1951 catgaggtgg catactggta cattctaagc attggagcac agactgactt2001 cctttctgtc ttcttctctg gatatacctt caaacacaaa atggtctatg2051 aagacacact caccctattc ccattctcag gagaaactgt cttcatgtcg2101 atggaaaacc caggtctatg gattctgggg tgccacaact cagactttcg2151 gaacagaggc atgaccgcct tactgaaggt ttctagttgt gacaagaaca2201 ctggtgatta ttacgaggac agttatgaag atatttcagc atacttgctg2251 agtaaaaaca atgccattga accaagaagc ttctcccaga attcaagaca2301 ccctagcact aggcaaaagc aatttaatgc caccacaatt ccagaaaatg2351 acatagagaa gactgaccct tggtttgcac acagaacacc tatgcctaaa2401 atacaaaatg tctcctctag tgatttgttg atgctcttgc gacagagtcc2451 tactccacat gggctatcct tatctgatct ccaagaagcc aaatatgaga2501 ctttttctga tgatccatca cctggagcaa tagacagtaa taacagcctg2551 tctgaaatga cacacttcag gccacagctc catcacagtg gggacatggt2601 atttacccct gagtcaggcc tccaattaag attaaatgag aaactgggga2651 caactgcagc aacagagttg aagaaacttg atttcaaagt ttctagtaca2701 tcaaataatc tgatttcaac aattccatca gacaatttgg cagcaggtac2751 tgataataca agttccttag gacccccaag tatgccagtt cattatgata2801 gtcaattaga taccactcta tttggcaaaa agtcatctcc ccttactgag2851 tctggtggac ctctgagctt gagtgaagaa aataatgatt caaagttgtt2901 agaatcaggt ttaatgaata gccaagaaag ttcatgggga aaaaatgtat2951 cgtcaacaga gagtggtagg ttatttaaag ggaaaagagc tcatggacct3001 gctttgttga ctaaagataa tgccttattc aaagttagca tctctttgtt3051 aaagacaaac aaaacttcca ataattcagc aactaataga aagactcaca3101 ttgatggccc atcattatta attgagaata gtccatcagt ctggcaaaat3151 atattagaaa gtgacactaa gtttaaaaaa gtgacacctt tgattcatga3201 cagaatgctt atggacaaaa atgctacagc tttgaggcta aatcatatgt3251 caaataaaac tacttcatca aaaaacatgg aaatggtcca acagaaaaaa3301 gagggcccca ttccaccaga tgcacaaaat ccagatatgt cgttctttaa3351 gatgctattc ttgccagaat cagcaaggtg gatacaaagg actcatggaa3401 agaactctct gaactctggg caaggcccca gtccaaagca attagtatcc3451 ttaggaccag aaadatctgt ggaaggtcag aatttcttgt ctgagaaaaa3501 caaagtggta gtaggaaagg gtgaatttac aaaggacgta ggactcaaag3551 agatggtttt tccaagcagc agaaacctat ttcttactaa cttggataat3601 ttacatgaaa ataatacata caatcaagaa aaaaaaattc aggaagaaat3651 agaaaagaag gaaacattaa tccaagagaa tgtagttttg cctcagatac3701 atacagtgac tggcactaag aatttcatga agaacctttt cttactgagc3751 actaggcaaa atgtagaagg ttcatatgac ggggcatatg ctccagcact3801 tcaagatttt aggtcattaa atgattcaac aaatagaaca aagaaacaca3851 cagctcattt ctcaaaaaaa ggggaggaag aaaacttgga aggcttggga3901 aatcaaacca agcaaattgt agagaaatat gcatgcacca caaggatatc3951 tcctaataca agccagcaga attttgtcac gcaacgtagt aagagagctt4001 tgaaacaatt cagactccca ctagaagaaa cagaacttga aaaaaggata4051 attgtggatg acacctcaac ccagtggtcc aaaaacatga aacatttgac4101 cccgagcacc ctcacacaga tagactacaa tgagaaggag aaaggggcca4151 ttactcagtc tcccttatca gattgcctta cgaggagtca tagcatccct4201 caagcaaata gatctccatt acccattgca aaggtatcat catctccatc4251 tattagacct atatatctga ccagggtcct attccaagac aactcttctc4301 atcttccagc agcatcttat agaaagaaag attctggggt ccaagaaagc4351 agtcatttct uacaaggagc caaaaaaaat aacctttctt tagccattct4401 aaccttggag atgactggtg atcaaagaga ggttggctcc ctggggacaa4451 gtgccacaaa ttcagtcaca tacaagaaag ttgagaacac tgttctcccg4501 aaaccagact tgcccaaaac atctggcaaa gttgaattgc ttccaaaagt4551 tcacatttat cagaaggacc tattccctac ggaaactagc aatgggtctc4601 ctggccatct ggatctcgtg gaagggagcc tucttcaggg aacagaggga4651 gcgattaagt ggaatgaagc aaacagacct ggaaaagttc cctttctgag4701 agtagcaaca gaaagctctg caaagactcc ctccaagcta ttggatcctc4751 ttgcttggga taaccactat ggtactcaga taccaaaaga agagtggaaa4801 tcccaagaga agtcaccaga aaaaacagct tttaagaaaa aggataccat4851 tttgtccctg aacgcttgtg aaagcaatca tgcaatagca gcaataaatg4901 agggacaaaa taagcccgaa atagaagtca cctgggcaaa gcaaggtagg4951 actgaaaggc tgtgctctca aaacccacca gtcttgaaac gccatcaacg5001 ggaaataact cgtactactc ttcagtcaga tcaagaggaa attgactatg5051 atgataccat atcagtrgaa atgaagaagg aagattttga catttatgat5101 gaggatgaaa atcagagccc ccgcagcttt caaaagaaaa cacgacacta5151 ttttattgct gcagtggaga ggctctggga ttatgggatg agtagctccc5201 cacatgttct aagaaacagg gctcagagtg gcagngcccc tcagttcaag5251 aaagttgttt tccaggaact tactgatggc tcctttactc agcccttata5301 ccgtggagaa ctaaatgaac atttgggact cctggggcca tatataagag5351 cagaagttga agataatanc atggtaactt tcagaaatca ggcctctcgt5401 ccctattcct tctattctag ccttatttct tatgaggaag atcagaggca5451 aggagcagaa cctagaaaaa actttgtcaa gcctaatgaa accaaaactt5501 acttttggaa agtgcaacat catatggcac ccactaaaga tgagtttgac5551 tgcaaagcct gggcttattt ctctgatgtt gacctggaaa aagatgtgca5601 ctcaggcctg attggacccc ttctggtctg ccacactaac acactgaacc5651 ctgctcatgg gagacaagcg acagtacagg aatttgctct gtttttcacc5701 atctttgatg agaccaaaag ctggtacttc actgaaaata tgqaaaqaaa5751 ctgcagggct ccctgcaata tccagatgga agatcccact tttaaagaga5801 attatcgctt ccatgcaatc aatggctaca taatggatac actacctggc5851 ttagtaatgg ctcaggatca aaggattcga tggtatctgc tcagcatggg5901 cagcaatgaa aacatccatt ctattcattt cagtggacat gtgttcactg5951 tacgaaaaaa agaggagtat aaaatggcac tgtacaatct ctatccaggt6001 gtttttgaga cagtggaaat gttaccatcc aaagctggaa tttggcgggt6051 ggaatgcctt attggcgagc atctacatgc tgggatgagc acactttttc6101 tggtgtacag caataagtgt cagactcccc tgggaatggc ttctggacac6151 attagagatt ttcagattac agcttcagga caatatggac agtgggcccc6201 aaagctggcc agacttcatt attccggatc aatcaatgcc tggagcacca6251 aggagccctt ttcttggatc aaggtggatc tgttggcacc aatgattatt6301 cacggcatca agacccaggg tgcccgtcag aagttctcca gcctctacat6351 ctctcagttt atcatcatgt atagtcttga tgggaagaag tggcagactt6401 atcgaggaaa ttccactgga accttaatgg tcttctttgg caatgtggat6451 tcatctggga taaaacacaa tatttttaac cctccaatta ttgctcgata6501 catccgtttg cacccaactc attatagcat tcgcagcact cttcgcatgg6551 agttgatggg ctgtgattta aatagttgca gcatgccatt gggaatggag6601 agtaaagcaa tatcagatgc acagattact gcttcatcct actttaccaa6651 tatgtttgcc acctggtctc cttcaaaagc tcgacttcac ctccaaggga6701 ggagtaatgc ctggagacct caggtgaata atccaaaaga gtggctgcaa6751 gtggacttcc agaagacaat gaaagtcaca ggagtaacta ctcagggagt6801 aaaatctctg cttaccagca tgtatgtgaa ggagttcctc atctccagca6851 gtcaagatgg ccatcagtgg actctctttt ttcagaatgg caaagtaaag6901 gtttttcagg gaaatcaaga ctccttcaca cctgtggtga actctctaga6951 cccaccgtta ctgactcgct accttcgaat tcacccccag agttgggtgc7001 accagattgc cctgaggatg gaggttctgg gctgcgaggc acaggacctc7051 tacgacaaaa ctcacacatg cccaccgtgc ccagctccag aactcctggg7101 cggaccgtca gtcttcctct tccccccaaa acccaaggac accctcatga7151 tctcccggac ccctgaggtc acatgcgtgg tggtggacgt gagccacgaa7201 gaccctgagg tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa7251 tgccaagaca aagccgcggg aggagcagta caacagcacg taccgtgtgg7301 tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac7351 aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat7401 ctccaaagcc aaagggcagc cccgagaacc acaggtgtac accctgcccc7451 catcccggga tgagctgacc aagaaccagg tcagcctgac ctgcctggtc7501 aaaggcttct atcccagcga catcgccgtg gagtgggaga gcaatgggca7551 gccggagaac aactacaaga ccacgcctcc cgtgttggac tccgacggct7601 ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag7651 gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta7701 cacgcagaag agcctctccc tgtctccggg taaaC. FVIII-Fc Heterodimer Hybrid(i) FVIII Heavy Chain (HC)-Fc DNA sequence (no linkerbetween HC and Fc) (signal peptide underlined, Fc region inbold) (SEQ ID NO: 7, which encodes SEQ ID NO: 8)

 101 actatatgca aagtgatctc ggtgagctgc ctgtggacgc aagatttcct 151 cctagagtgc caaaatcttt tccattcaac acctcagtcg tgtacaaaaa 201 gactctgttt gtagaattca cggatcacct tttcaacatc gctaagccaa 251 ggccaccctg gatgggtctg ctaggtccta ccatccaggc tgaggtttat 301 gatacagtgg tcattacact taagaacatg gcttcccatc ctgtcagtct 351 tcatgctgtt ggtgtatcct actggaaagc ttctgaggga gctgaatatg 401 atgatcagac cagtcaaagg gagaaagaag atgataaagt cttccctggt 451 ggaagccata catatgtctg gcaggtcctg aaagagaatg gtccaatggc 501 ctctgaccca ctgtgcctta cctactcata tctttctcat gtggacctgg 551 taaaagactt gaattcaggc ctcattggag ccctactagt atgtagagaa 601 gggagtctgg ccaaggaaaa gacacagacc ttgcacaaat ttatactact 651 ttttgctgta tttgatgaag ggaaaagttg gcactcagaa acaaagaact 701 ccttgatgca ggatagggat gctgcatctg ctcgggcctg gcctaaaatg 751 cacacagtca atggttatgt aaacaggtct ctgccaggtc tgattggatg 801 ccacaggaaa tcagtctatt ggcatgtgat tggaatgggc accactcctg 851 aagtgcactc aatattcctc gaaggtcaca catttcttgt gaggaaccat 901 cgccaggcgt ccttggaaat ctcgccaata actttcctta ctgctcaaac 951 actcttgatg gaccttggac agtttctact gttttgtcat atctcttccc1001 accaacatga tggcatggaa gcttatgtca aagcagacag ctgtccagag1051 gaaccccaac tacgaatgaa aaataatgaa gaagcggaag actatgatga1101 tgatcttact gattctgaaa tggatgtggt caggtttgat gatgacaact1151 ctccttcctt tatccaaatt cgctcagttg ccaagaagca tcctaaaact1201 tgggtacatt acattgctgc tgaagaggag gactgggact atgctccctt1251 agtcctcgcc cccgatgaca gaagttataa aagtcaatat ttgaacaatg1301 gccctcagcg gattggtagg aagtacaaaa aagtccgatt tatggcatac1351 acagatgaaa cctttaagac tcgtgaagct attcagcatg aatcaggaat1401 cttgggacct tractttatg gggaagttgg agacacactg ttgattatat1451 ttaagaatca agcaagcaga ccatataaca tctaccctca cggaatcact1501 gatgtccgtc ctttgtattc aaggagatta ccaaaaggtg taaaacattt1551 gaaggatttt ccaattctgc caggagaaat attcaaatat aaatggacag1601 tgactgtaga agatgggcca actaaatcag atcctcggtg cctgacccgc1651 tattactcta gtttcgttaa tatggagaga gatctagctt caggactcat1701 tggccctctc ctcatctgct acaaagaatc tgtagatcaa agaggaaacc1751 agataatgtc agacaagagg aatgtcatcc tgttttctgt atttgatgag1801 aaccgaagct ggtacctcac agagaatata caacgctttc tccccaatcc1851 agctggagtg cagcttgagg atccagagtt ccaagcctcc aacatcatgc1901 acagcatcaa tggctatgtt tttgatagtt tgcagttgtc agtttgtttg1951 catgaggtgg catactggta cattctaagc attggagcac agactgactt2001 cctttctgtc ttcttctctg gatatacctt caaacacaaa atggtctatg2051 aagacacact caccctattc ccattctcag gagaaactgt cttcatgtcg2101 atggaaaacc caggtctatg gattctgggg tgccacaact cagactttcg2151 gaacagaggc atgaccgcct tactgaaggt ttctagttgt gacaagaaca2201 ctggtgatta ttacgaggac agttatgaag atatttcagc atacttgctg2251 agtaaaaaca atgccattga accaagagac aaaactcaca catgcccacc2301 gtgcccagct ccagaactcc tgggcggacc gtcagtcttc ctcttccccc2351 caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacatgc2401 gtggtggtgg acgtgagcca cgaagaccct gaggtcaagt tcaactggta2451 cgtggacggc gtggaggtgc ataatgccaa gacaaagccg cgggaggagc2501 agtacaacag cacgtaccgt gtggtcagcg tcctcaccgt cctgcaccag2551 gactggctga atggcaagga gtacaagtgc aaggtctcca acaaagccct2601 cccagccccc atcgagaaaa ccatctccaa agccaaaggg cagccccgag2651 aaccacaggt gtacaccctg cccccatccc gggatgagct gaccaagaac2701 caggtcagcc tgacctgcct ggtcaaaggc ttctatccca gcgacatcgc2751 cgtggagtgg gagagcaatg ggcagccgga gaacaactac aagaccacgc2801 ctcccgtgtt ggactccgac ggctccttct tcctctacag caagctcacc2851 gtggacaaga gcaggtggca gcaggggaac gtcttctcat gctccgtgat2901 gcatgaggct ctgcacaacc actacacgca gaagagcctc tccctgtctc2951 cgggtaaa (ii) FVIII Heavy Chain (HC)-Fc DNA sequence (5 amino acidlinker between HC and Fc) (signal peptide underlined, Fcregion in bold, 5 amino acid linker is double-underlined) (SEQ ID NO: 9, which encodes SEQ ID NO: 10)

 101 actatatgca aagtgatctc ggtgagctgc ctgtggacgc aagatttcct 151 cctagagtgc caaaatcttt tccattcaac acctcagtcg tgtacaaaaa 201 gactctgttt gtagaattca cggatcacct tttcaacatc gctaagccaa 251 ggccaccctg gatgggtctg ctaggtccta ccatccaggc tgaggtttat 301 gatacagtgg tcattacact taagaacatg gcttcccatc ctgtcagtct 351 tcatgctgtt ggtgtatcct actggaaagc ttctgaggga gctgaatatg 401 atgatcagac cagtcaaagg gagaaagaag atgataaagt cttccctggt 451 ggaagccata catatgtctg gcaggtcctg aaagagaatg gtccaatggc 501 ctctgaccca ctgtgcctta cctactcata tctttctcat gtggacctgg 551 taaaagactt gaattcaggc ctcattggag ccctactagt atgtagagaa 601 gggagtctgg ccaaggaaaa gacacagacc ttgcacaaat ttatactact 651 ttttgctgta tttgatgaag ggaaaagttg gcactcagaa acaaagaact 701 ccttgatgca ggatagggat gctgcatctg ctcgggcctg gcctaaaatg 751 cacacagtca atggttatgt aaacaggtct ctgccaggtc tgattggatg 801 ccacaggaaa tcagtctatt ggcatgtgat tggaatgggc accactcctg 851 aagtgcactc aatattcctc gaaggtcaca catttcttgt gaggaaccat 901 cgccaggcgt ccttggaaat ctcgccaata actttcctta ctgctcaaac 951 actcttgatg gaccttggac agtttctact gttttgtcat atctcttccc1001 accaacatga tggcatggaa gcttatgtca aagtagacag ctgtccagag1051 gaaccccaac tacgaatgaa aaataatgaa gaagcggaag actatgatga1101 tgatcttact gattctgaaa tggatgtggt caggtttgat gatgacaact1151 ctccttcctt tatccaaatt cgctcagttg ccaagaagca tcctaaaact1201 tgggtacatt acattgctgc tgaagaggag gactgggact atgctccctt1251 agtcctcgcc cccgatgaca gaagttataa aagtcaatat ttgaacaatg1301 gccctcagcg gattggtagg aagtacaaaa aagtccgatt tatggcatac1351 acagatgaaa cctttaagac tcgtgaagct attcagcatg aatcaggaat1401 cttgggacct ttactttatg gggaagttgg agacacactg ttgattatat1451 ttaagaatca agcaagcaga ccatataaca tctaccctca cggaatcact1501 gatgtccgtc ctttgtattc aaggagatta ccaaaaggtg taaaacattt1551 gaaggatttt ccaattctgc caggagaaat attcaaatat aaatggacag1601 tgactgtaga agatgggcca actaaatcag atcctcggtg cctgacccgc1651 tattactcta gtttcgttaa tatggagaga gatctagctt caggactcat1701 tggccctctc ctcatctgct acaaagaatc tgtagatcaa agaggaaacc1751 agataatgtc agacaagagg aatgtcatcc tgttttctgt atttgatgag1801 aaccgaagct ggtacctcac agagaatata caacgctttc tccccaatcc1851 agctggagtg cagcttgagg atccagagtt ccaagcctcc aacatcatgc1901 acagcatcaa tggctatgtt tttgatagtt tgcagttgtc agtttgtttg1951 catgaggtgg catactggta cattctaagc attggagcac agactgactt2001 cctttctgtc ttcttctctg gatatacctt caaacacaaa atggtctatg2051 aagacacact caccctattc ccattctcag gagaaactgt cttcatgtcg2101 atggaaaacc caggtctatg gattctgggg tgccacaact cagactttcg2151 gaacagaggc atgaccgcct tactgaaggt ttctagttgt gacaagaaca2201 ctggtgatta ttacgaggac agttatgaag atatttcagc atacttgctg

2301 tcacacatgc ccaccgtgcc cagctccaga actcctgggc ggaccgtcag2351 tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc2401 cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt2451 caagttcaac tggtacgtgg acggcgtgga ggtgcataat gccaagacaa2501 agccgcggga ggagcagtac aacagcacgt accgtgtggt cagcgtcctc2551 accgtcctgc accaggactg gctgaatggc aaggagtaca agtgcaaggt2601 ctccaacaaa gccctcccag cccccatcga gaaaaccatc tccaaagcca2651 aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggat2701 gagctgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta2751 tcccagcgac atcgccgtgg agtgggagag caatgggcag ccggagaaca2801 actacaagac cacgcctccc gtgttggact ccgacggctc cttcttcctc2851 tacagcaagc tcaccgtgga caagagcagg tggcagcagg ggaacgtctt2901 ctcatgctcc gtgatgcatg aggctctgca caaccactac acgcagaaga2951 gcctctccct gtctccgggt aaa(iii) FVII1 Light Chain (LC)-Fc DNA sequence (signal peptideunderlined, Fc region in bold) (SEQ ID NO: 11, which encodesSEQ ID NO: 12)

 101 ttgactatga tgataccata tcagttgaaa tgaagaagga agattttgac 151 atttatgatg aggatgaaaa tcagagcccc cgcagctttc aaaagaaaac 201 acgacactat tttattgctg cagtggagag gctctgggat tatgggatga 251 gtagctcccc acatgttcta agaaacaggg ctcagagtgg cagtgtccct 301 cagttcaaga aagttgtttt ccaggaattt actgatggct cctttactca 351 gcccttatac cgtggagaac taaatgaaca tttgggactc ctggggccat 401 atataagagc agaagttgaa gataatatca tggtaacttt cagaaatcag 451 gcctctcgtc cctattcctt ctattctagc cttatttctt atgaggaaga 501 tcagaggcaa ggagcagaac ctagaaaaaa ctttgtcaag cctaatgaaa 551 ccaaaactta cttttggaaa gtgcaacatc atatggcacc cactaaagat 601 gagtttgact gcaaagcctg ggcttatttc tctgatgttg acctggaaaa 651 agatgtgcac tcaggcctga ttggacccct tctggtctgc cacactaaca 701 cactgaaccc tgctcatggg agacaagtga cagtacagga atttgctctg 751 tttttcacca tctttgatga gactaaaagc tugtacttca ctgaaaatat 801 ggaaagaaac tgcagggctc cctgcaatat ccagatggaa gatcccactt 851 ttaaagagaa ttatcgcttc catgcaataa atggctacat aatggataca 901 ctacctggct tagtaatggc tcaggatcaa aggattcgat gatatctgat 951 cagaatggga agtaatgaaa acatatattc tattcatttc agtggaaatg1001 tgttcattgt acgaaaaaaa gaggagtata aaatggcact gtacaatctc1051 tatccaggtg tttttgagac agtggaaatg ttatcatcca aagctggaat1101 ttggtgggtg gaatgcctta ttggcgagca tctacatgat gggatgagca1151 cattttttct ggtgtacagc aataagtgtc agattcacct gggaatggct1201 tctggacaca ttagagattt tcagattaca gcttcaggac aatatggaca1251 gtgggcccca aagctggcca gacttcatta ttccggatca atcaatgcct1301 ggagcaccaa ggagcccttt tcttggatca aggtagatct gttggcacca1351 atgattattc acggcatcaa gacccagggt gcccgtcaga agttctccag1401 cctctacatc tctcagttta tcatcatgta tagtcttgat gggaagaagt1451 ggcagaatta tagaggaaat tccactggaa ccttaatggt cttctttggc1501 aatgtggatt catctgggat aaaacacaat atttttaacc ctccaattat1551 tgctcgatac atccgtttgc acccaactca ttatagcatt cgcagcactc1601 ttcgcatgga gttgatgggc tgtgatttaa atagttgcag catgccattg1651 ggaatggaga gtaaagcaat atcagatgca cagattactg cttcatccta1701 ctttaccaat atgtttgcca cctggtctcc ttcaaaagct cgacttcacc1751 tccaagggag gagtaatgcc tggagacctc aggtgaataa tccaaaagag1301 tggctgcaag tggacttcca gaagacaatg aaagtcacag gagtaactac1351 tcagggagta aaatctctgc ttaccagcat gtatgtgaag gagttcctca1901 tctccagcag tcaagatggc catcagtgga ctctcttttt tcagaatggc1951 aaagtaaagg tttttcaggg aaatcaagac tccttcacac ctgtggtgaa2001 ctctctagac ccaccgttac tgactcgcta ccttcgaatt cacccccaga2051 gttgggtgca ccagattgcc ctgaggatgg aggttctggg ctgcgaggca2101 caggacctct acgacaaaac tcacacatgc ccaccgtgcc cagctccaga2151 actcatgggc ggaccgtcag tcttcctctt cccxc.aaaa cccaaggaca2201 ccctcatgat ctcccggacc cctgaggtca catgcgtggt ggtggacgtg2251 agccacgaag accctgaggt caagttcaac tggtacgtgg acggcgtgga2301 ggtgcataat gccaagacaa agccgcggga ggagcagtac aacagcacgt2351 accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc2401 aaggagtaca agtgcaaggt ctccaacaaa gccctcccag cccccatcga2451 gaaaaccatc tccaaagcca aagggcagcc ccgagaacca caggtgtaca2501 ccctgccccc atcccgggat gagctgacca agaaccaggt cagcctgacc2551 tgcctggtca aaggcttcta tcccagcgac atcgccgtgg agtgggagag2601 caatgggcag ccggagaaca actacaagac cacgcctccc gtgttggact2651 ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg2701 tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca2751 caaccactac acgcagaaga gcctctccct gtctccgggt aaa

TABLE 16 Polypeptide Sequences of FVIIIA. B-Domain Deleted FVIII-Fc Monomer Hybrid (BDD FVIIIFcmonomer dimer): created by coexpressing BDD FVIIIFc and Fc chains.Construct = HC-LC-Fc fusion. An Fc expression cassette iscotransfected with BDDFVIII-Fc to generate the BDD FVIIIFcmonomer-. For the BDD FVIIIFc chain, the Fc sequence isshown in bold; HC sequence is shown in double underline;remaining B domain sequence is shown in italics. Signalpeptides are underlined.i) B domain deleted FVIII-Fc chain (19 amino acid signalsequence underlined) (SEQ ID NO: 2)

 801 DFDIYDEDEN QSPRSFQKKT RHYFIAAVER LWDYGMSSSP HVLRNRAQSG 851 SVPQFKKVVF QEFTDGSFTQ PLYRGELNEH LGLLGPYIRA EVEDNIMVTF 901 RNQASRPYSF YSSLISYEED QRQGAEPRKN FVKPNETKTY FWKVQHHMAP 951 TKDEFDCKAW AYFSDVDLEK DVHSGLIGPL LVCHTNTLNP AHGRQVTVQE1001 FALFFTIFDE TKSWYFTENM ERNCRAPCNI QMEDPTFKEN YRFHAINGYI1051 MDTLPGLVMA QDQRIRWYLL SMGSNENIHS IHFSGHVFTV RKKEEYKMAL1101 YNLYPGVFET VEMLPSKAGI WRVECLIGEH LHAGMSTLFL VYSNKCQTPL1151 GMASGHIRDF QITASGQYGQ WAPKLARLHY SGSINAWSTK EPFSWIKVDL1201 LAPMIIHGIK TQGARQKFSS LYISQFIIMY SLDGKKWQTY RGNSTGTLMV1251 FFGNVDSSGI KHNIFNPPII ARYIRLHPTH YSIRSTLRME LMGCDLNSCS1301 MPLGMESKAI SDAQITASSY FTNMFATWSP SKARLHLQGR SNAWRPQVNN1351 PKEWLQVDFQ KTMKVTGVTT QGVKSLLTSM YVKEFLISSS QDGHQWTLFF1401 QNGKVKVFQG NQDSFTPVVN SLDPPLLTRY LRIHPQSWVH QTAIRMEVLG1451 CEAQDLYDKT HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV1501 VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV SVLTVLHQDW1551 LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP SRDELTKNQV1601 SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD1651 KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGKii) Fc chain (20 amino acid heterologous signal peptide from mouse Igκchain underlined) (SEQ ID NO: 4)

  51 LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY 101 RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PLEKTISKAK GQPREPQVYT 151 LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS 201 DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGKB. Full length FVIIIFc monomer hybrid (Full length FVIIIFcmonomer dimer): created by coexpressing FVIIIFc and Fc chains.Construct = HC-B-LC-Fc fusion. An Fc expression cassette iscotransfected with full length FVIII-Fc to generate the fulllength FVIIIFc monomer. For the FVIIIFc chain, the Fcsequence is shown in bold; HC sequence is shown in doubleunderline; B domain sequence is shown in italics. Signalpeptides are underlined.i) Full length FVIIIFc chain (FVIII signal peptideunderlined (SEQ ID NO: 6)

 801 IQNVSSSDLL MLLRQSPTPH GLSLSDLQEA KYETFSDDPS PGAIDSNNSL 851 SEMTHERPQL HHSGDMVFTP ESGLQLRLNE KLGTTAATEL KKLDFKVSST 901 SNNLISTIPS DNLAAGTDNT SSLGPPSMPV HYDSQLDTTL FGKKSSPLTE 951 SGGPLSLSEE NNDSKLLESG LMNSQESSWG KNVSSTESGR LFWGKRAHGP1001 ALLTKDNALF KVSISLLKTN KTSNNSATNR KTHIDGPSLL IENSPSVWQN1051 ILESDTEFEK VTPLIHDRML MDKNATALRL NRMSNKTTSS KNMEMVQQKK1101 EGPIPPDAQN PDMSFTKMLF LPESARWIQR THGKNSLNSG QGPSPKQLVS1151 LGPEKSVEGQ NTLSEKNKVV VGKGEFTKDV GLKEMVFPSS RNLFLTNLDN1201 LHENNTHNQE KKIQEEIEKK ETLIQENVVL PQIHTVTGTK NFMKNLFILS1251 TRQNVEGSYD GAYAPVLQDF RSLNDSTNRT KKHTAHFSKK GEEENLEGLG1301 NQTKQIVEKY ACTTRISPNT SQQNFVTQRS KRALKQFRLP LEETELEKRI1351 IVDDTSTQWS KNMKHLTPST LTQIDYNEKE KGAITQSPLS DCLTRSESIP1401 QANRSPLPIA KVSSFPSIRP TYLTRVLFQD NSSHLPAASY RKWDSGVQES1451 SHFLQGAKKN NLSLAILTLE MTGDQREVGS LGTSATNSVT YKKVENTVLP1501 KPDLPKTSGK VELLPKVHIY QKDLPPTETS NGSPGHLDLV EGSLLQGTEG1551 AIKWNEANRP GKVPFLRVAT ESSAKTPSKL LDPLAWDNHY GTQIPKEEWK1601 SQEKSPEKTA FKKKDTILSL NACESNHAIA AINEGQNKPE IEVTWAKQGR1651 TERLCSQNPP VLKRHQREIT RTTLQSDQEE IDYDDTISVE MKKEDFDIYD1701 EDENQSPRSF QKKTRHYFIA AVERLWDYGM SSSPHVLRNR AQSGSVPQFK1751 KVVFQEFTDG SFTQPLYRGE LNEHLGLLGP YIRAEVEDNI MVTFRNQASR1801 PYSFYSSLIS YEEDQRQGAE PRKNFVKPNE TKTYFWKVQH HMAPTKDEFD1851 CKAWAYESDV DLEKDVHSGL IGPLLVCHTN TLNPAHGRQV TVQEFALFFT1901 IFDETKSWYF TENMERNCRA PCNIQMEDPT FKENYRFHAI NGYIMDTLPG1951 LVMAQDQRIR WYLLSMGSNE NIHSIHFSGH VFTVRKKEEY KMALYNLYPG2001 VFETVEMLPS KAGIWRVECL IGEHLHAGMS TLFLVYSNKC QTPLGMASGH2051 IRDFQITASG QYGQWAPKLA RLHYSGSINA WSTKEPFSWI KVDLLAPMII2101 HGIKTQGARQ KFSSLYISQF IIMYSLDGKK WQTYRGNSTG TLMVFFGNVD2151 SSGIKHNIFN PPIIARYIRL HPTHYSIRST LRMELMGCDL NSCSMPLGME2201 SKAISDAQIT ASSYFTNMFA TWSPSKARLH LQGRSNAWRP QVNNPKEWLQ2251 VDFQKTMKVT GVTTQGVKSL LTSMYVKEFL ISSSQDGHQW TLFFQNGKVK2301 VFQGNQDSFT PVVNSLDPPL LTRYLRIHPQ SWVHQIALRM EVLGCEAQDL2351 YDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSRE2401 DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY2451 KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV2501 KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ2551 GNVFSCSVMH EALHNHYTQK SLSLSPGK C. FVIII-Fc Heterodimer HybridThis is made by cotransfecting HC-Fc and LC-Fc constructs.Two HC-Fc constructs have been made. One has no linkerbetween HC and Fc (HC-Fc) while the other has a 5 amino acidlinker between HC and Fc (HC+5-Fc). The FVIII signal peptidewas used for the HC-Fc constructs, while the mouse Igκ signalsequence was used for the LC-Fc construct.(i) HC-Fc (Fc sequence is shown in bold, signal peptideunderlined) (SEQ ID NO: 8)

  51 PRVPKSFPFN TSVVYKKTLF VEFTDHLFN1 AKPRPPWMGL LGPTIQAEVY 101 DTVVITLKNM ASHPVSLHAV GVSYWKASEG AEYDDQTSQR EKEDDKVFPG 151 GSHTYVWQVL KENGPMASDP LCLTYSYLSH VDLVKDLNSG LIGALLVCRE 201 GSLAKEKTQT LRKFILLFAV FDEGKSwHSE TKNSLMQDRD AASARAWPKM 251 HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL EGHTFLVRNH 301 RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME AYVKVDSCPE 351 EPQLRMKNNE EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI RSVAKKHPKT 401 WVHYIAAEEE DWDYAPLVLA PDDRSYKSQY LNNGPQRIGR KYKKVRFMAY 451 TDETFKTREA IQHESGILGP LLYGEVGDTL LIIFKNQASR PYNIYPHGIT 501 DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP TKSDPRCLTR 551 YYSSFVNMER DLASGLIGPL LIGYKESVDQ RGNQIMSDKR NVILFSVFDE 601 NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHSINGYV FDSLQLSVCL 651 HEVAYWYILS IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF PFSGETVFMS 701 MENPGLWILG CHNSDFRNRG MTALLKVSSC DKNTGDYYED SYEDISAYLL 751 SKNNAYEPRD KTHTCPPCPA PELLGGPSVF LFPPKPKDTL MISRTPEVTC 801 VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYNSTYR VVSVLTVLHQ 851 DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTL PPSRDELTKN 901 QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTIPPVLDSD GSFFLYSKLT 951 VDKSRWQQGN VPSCSVMHEA LHNHYTQKSL SLSPGK(ii) HC+5-Fc (Fc sequence is shown in bold, 5 amino acidlinker sequence (from the B domain of FVIII) is shown initalics, signal peptide underlined.) (SEQ ID NO: 10)

  51 PRVPKSFPFN TSVVYKKTLF VEFTDHLFN1 AKPRPPWMGL LGPTIQAEVY 101 DTVVITLKNM ASHPVSLHAV GVSYWKASEG AEYDDQTSQR EKEDDKVFPG 151 GSHTYVWQVL KENGPMASDP LCLTYSYLSH VDLVKDLNSG L1GALLVCRE 201 GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD AASARAWPKM 251 HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL EGHTFLVRNH 301 RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME AYVKVDSCPE 351 EPQLRMKNNE EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI RSVAKKHPKT 401 WVHYIAAEEE DWDYAPLVLA PDDRSYKSQY LNNGPQRIGR KYKKVRFMAY 451 TDETFKTREA IQHESGILGP LLYGEVGDTL LIIFKNQASR PYNTYPHGIT 501 DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP TKSDPRCLTR 551 YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR NVILFSVFDE 601 NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHSINGYV FDSLQLSVCL 651 HEVAYWYILS IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF PFSGETVFMS 701 MENPGLWILG CHNSDFRNRG MTALLKVSSC DKNTGDYYED SYEDISAYLL 751 SKNNAIEPRS FSQN DKTHTC PPCPAPELLG GPSVFLFPPK PKDTLMISRT 801 PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN AKTKPREEQY NSTYRVVSVL 851 TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREP QVYTLPPSRD 901 ELTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP VLDSDGSFPL 951 YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG K(iii) LC-Fc6His (Fc sequence is shown in bold, signalpeptide underlined.) (SEQ ID NO: 12)

  51 IYDEDENQSP RSFQKKTRHY FIAAVERLWD YGMSSSPHVL RNRAQSGSVP 101 QFKKVVFQEF TDGSFTQPLY RGELNEHLGL LGPYIRAEVE DNIMVTFRNQ 151 ASRPYSFYSS LISYEEDQRQ GAEPRKNFVK PNETKTYFWK VQHHMAPTKD 201 EFDCKAWAYF SDVDLEKDVH SGLIGPLLVC HTNTLNPAHG RQVTVQEFAL 251 FFTIFDETKS WYFTENMERN CRAPCNIQME DPTFKENYRF HAINGYIMDT 301 LPGLVMAQDQ RIRWYLLSMG SNENTHSIHF SGHVFTVRKK EEYKMALYNL 351 YPGVFETVEM LPSKAGiWRV ECLIGEHLHA GMSTLFLVYS NKCQTPLGMA 401 SGHIRDFQIT ASGQYGQWAP KLARLHYSGS INAWSTKEPF SWIKVDLLAP 451 MIIHGIKTQG ARQKFSSLYI SQFIIMYSLD GKKWQTYRGN STGTLMVFFG 501 NVDSSGIKHN IFNPPTIARY IRLHPTHYSI RSTLRMELMG CDLNSCSMPL 551 GMESKAISDA QITASSYFTN MFATWSPSKA RLHLQGRSNA WRPQVNNPKE 601 WLQVDFQKTM KVTGVTTQGV KSLLTSMYVK EFLISSSQDG HQWTLFFQNG 651 KVKVFQGNQD SFTPVVNSLD PPLLTRYLRI HPQSWVHQTA LRMEVLGCEA 701 QDLYDKTHTC PPCPAPELLG GPSVFLFPPK PKDTLMISRT PEVTCVVVDV 751 SHEDPEVKFN WYVDGVEVHN AKTKPREEQY NSTYRVVSVL TVLHQDWLNG 801 KEYKCKVSNK ALPAPIEKTI SKAKGQPREP QVYTLPPSRD ELTENQVSLT 851 CLVKGFYPSD IAVEWESNGQ PENNYKTIPP VLDSDGSFFL YSKLTVDKSR 901 WQQGNVFSCS VMHEALHNHY TQKSLSLSPG K

TABLE 17 Polynucleotide Sequences of FIXFIX-Fc Chain DNA Sequence (FIX signal peptide underlined,FIX sequence double underlined, Fc region in bold) (SEQ IDNO: 13, which encodes SEQ ID NO: 14)pSYN-FIX-030 Nucleotide sequence (nt 1 to 7583):FIX exon 1 (signal peptide, 1st amino acid propeptide): nt 690-777FIX mini intron: nt 778-1076 FIX sequence: nt 1077-2371 Fc: nt 2372-3052   1 gcgcgcgttg acattgatta ttgactagtt attaatagta atcaattacg  51 gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac 101 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt 151 caataatgac gtatgttccc atagtaacgc caatagggac tttccattga 201 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca 251 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat 301 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact 351 tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt 401 ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc 451 caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat 501 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat 551 gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctctctggc 601 taactagaga acccactgct tactggctta tcgaaattaa tacgactcac

 801 attgagtatg cttgcctttt agatatagaa atatctgatg ctgtcttctt 851 cactaaattt tgattacatg atttgacagc aatattgaag agtctaacag 901 ccagcacgca ggttggtaag tactgtggga acatcacaga ttttggctcc 951 atgccctaaa gagaaattgg ctttcagatt atttggatta aaaacaaaga1001 ctttcttaag agatgtaaaa ttttcatgat gttttctttt ttgctaaaac

2401 agctccggaa ctcctgggcg gaccgtcagt cttcctcttc cccccaaaac2451 ccaaggacac cctcatgatc tcccggaccc ctgaggtcac atgcgtggtg2501 gtggacgtga gccacgaaga ccctgaggtc aagttcaact ggtacgtgga2551 cggcgtggag gtgcataatg ccaagacaaa gccgcgggag gagcagtaca2601 acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg2651 ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc2701 ccccatcgag aaaaccatct ccaaagccaa agggcagccc cgagaaccac2751 aggtgtacac cctgccccca tcccgggatg agctgaccaa gaaccaggtc2801 agcctgacct gcctggtcaa aggcttctat cccagcgaca tcgccgtgga2851 gtgggagagc aatgggcagc cggagaacaa ctacaagacc acgcctcccg2901 tgttggactc cgacggctcc ttcttcctct acagcaagct caccgtggac2951 aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga3001 ggctctgcac aaccactaca cgcagaagag cctctccctg tctccgggta3051 aatgagaatt cagacatgat aagatacatt gatgagtttg gacaaaccac3101 aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta3151 ttgctttatt tgtaaccatt ataagctgca ataaacaagt tggggtgggc3201 gaagaactcc agcatgagat ccccgcgctg gaggatcatc cagccggcgt3251 cccggaaaac gattccgaag cccaaccttt catagaaggc ggcggtggaa3301 tcgaaatctc gtagcacgtg tcagtcctgc tcctcggcca cgaagtgcac3351 gcagttgccg gccgggtcgc gcagggcgaa ctcccgcccc cacggctgct3401 cgccgatctc ggtcatggcc ggcccggagg cgtcccggaa gttcgtggac3451 acgacctccg accactcggc gtacagctcg tccaggccgc gcacccacac3501 ccaggccagg gtgttgtccg gcaccacctg gtcctggacc gcgctgatga3551 acagggtcac gtcgtcccgg accacaccgg cgaagtcgtc ctccacgaag3601 tcccgggaga acccgagccg gtcggtccag aactcgaccg ctccggcgac3651 gtcgcgcgcg gtgagcaccg gaacggcact ggtcaacttg gccatggttt3701 agttcctcac cttgtcgtat tatactatgc cgatatacta tgccgatgat3751 taattgtcaa cacgtgctga tcagatccga aaatggatat acaagctccc3801 gggagctttt tgcaaaagcc taggcctcca aaaaagcctc ctcactactt3851 ctggaatagc tcagaggcag aggcggcctc ggcctctgca taaataaaaa3901 aaattagtca gccatggggc ggagaatggg cggaactggg cggagttagg3951 ggcgggatgg gcggagttag gggcgggact atggttgctg actaattgag4001 atgcatgctt tgcatacttc tgcctgctgg ggagcctggg gactttccac4051 acctggttgc tgactaattg agatgcatgc tttgcatact tctgcctgct4101 ggggagcctg gggactttcc acaccctcgt cgagctagct tcgtgaggct4151 ccggtgcccg tcagtgggca gagcgcacat cgcccacagt ccccgagaag4201 ttggggggag gggtcggcaa ttgaaccggt gcctagagaa ggtggcgcgg4251 ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg4301 gtgggggaga accgtatata agtgcagtag tcgccgtgaa cgttcttttt4351 cgcaacgggt ttgccgccag aacacaggta agtgccgtgt gtggttcccg4401 cgggcctggc ctctttacgg gttatggccc ttgcgtgcct tgaattactt4451 ccacctggct ccagtacgtg attcttgatc ccgagctgga gccaggggcg4501 ggccttgcgc tttaggagcc ccttcgcctc gtgcttgagt tgaggcctgg4551 cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg4601 tctcgctgct ttcgataagt ctctagccat ttaaaatttt tgatgacctg4651 ctgcgacgct ttttttctgg caagatagtc ttgtaaatgc gggccaggat4701 ctgcacactg gtatttcggt ttttggggcc gcgggcggcg acggggcccg4751 tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac4801 cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct4851 ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg4901 gtcggcacca gttgcgtgag cggaaagatg gccgcttccc ggccctgctc4951 cagggggctc aaaatggagg acgcggcgct cgggagagcg ggcgggtgag5001 tcacccacac aaaggaaagg ggcctttccg tcatcagccg tcgcttcatg5051 tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctgga5101 gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg5151 gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca5201 cttgatgtaa ttctccttgg aatttgccct ttttgagttt ggatcttggt5251 tcattctcaa gcctcagaca gtggttcaaa gtttttttct tccatttcag5301 gtgtcgtgaa cacgtggtcg cggccgcgcc gccaccatgg agacagacac5351 actcctgcta tgggtactgc tgctctgggt tccaggttcc actggtgaca5401 aaactcacac atgcccaccg tgcccagcac ctgaactcct gggaggaccg5451 tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg5501 gacccctgag gtcacatgcg tggtggtgga cgtgagccac gaagaccctg5551 aggtcaagtt caactggtac gtggacggcg tggaggtgca taatgccaag5601 acaaagccgc gggaggagca gtacaacagc acgtaccgtg tggtcagcgt5651 cctcaccgtc ctgcaccagg actggctgaa tggcaaggag tacaagtgca5701 aggtctccaa caaagccctc ccagccccca tcgagaaaac catctccaaa5751 gccaaagggc agccccgaga accacaggtg tacaccctgc ccccatcccg5801 cgatgagctg accaagaacc aggtcagcct gacctgcctg gtcaaaggct5851 tctatcccag cgacatcgcc gtggagtggg agagcaatgg gcagccggag5901 aacaactaca agaccacgcc tcccgtgttg gactccgacg gctccttctt5951 cctctacagc aagctcaccg tggacaagag caggtggcag caggggaacg6001 tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag6051 aagagcctct ccctgtctcc gggtaaatga ctcgagagat ctggccggct6101 gggcccgttt cgaaggtaag cctatcccta accctctcct cggtctcgat6151 tctacgcgta ccggtcatca tcaccatcac cattgagttt aaacccgctg6201 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct6251 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc6301 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat6351 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca6401 atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa6451 agaaccagtg gcggtaatac ggttatccac agaatcaggg gataacgcag6501 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag6551 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca6601 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa6651 gataccaggc gtttccccct agaagctccc tcgtgcgctc tcctgttccg6701 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt6751 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg6801 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc6851 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga6901 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt6951 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac7001 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttac tt7051 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta7101 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga7151 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa7201 cgaaaactca cgttaaggga ttttggtcat gacattaacc tataaaaata7251 ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa7301 aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc7351 ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg7401 ggtgtcgggg ctggcttaac tatgcggcat cagagcagat tgtactgaga7451 gtgcaccata tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa7501 taccgcatca ggcgccattc gccattcagg ctgcgcaact gttgggaagg7551 gcgatcggtg cgggcctctt cgctattacg cca

TABLE 18 Polypeptide Sequences of FIXFIX-Fc Monomer Hybrid: created by coexpressing FIX-Fc and Fc chains.A. FIX-Fc chain (46 amino acid signal sequence underlined)(SEQ ID NO: 14)The c-terminal lysine is not present in either subunit; thisprocessing is often observed in recombinant proteins producedin mammalian cell culture, as well as with plasma derived proteins.FIX-Fc-SC Subunit (the Fc part of FIX-Fc is in bold):

  51 KLEEFVQGNL EREEMEEKCS FEEAREVFEN TERTTEFWKQ YVDGDQCESN 101 PCLNGGSCKD DINSYECWCP FGFEGKNCEL DVTCNIKNGR CEQFCKNSAD 151 NKVVCSCTEG YRLAENQKSC EPAVPFPCGR VSVSQTSKLT RAETVFPDVD 201 YVNSTEAETI LDNITQSTQS ENDFTRVVGG EDAKPGQFPW QVVLNGKVDA 251 FCGGSIVNEK WIVTAAHCVE TGVKITVVAG EHNIEETEHT EQKRNVIRII 301 PHHNYNAAIN KYNHDIALLE LDEPLVLNSY VTPICIADKE YTNIFLKFGS 351 GYVSGWGRVF HKGRSALVLQ YLRVPLVDRA TCLRSTKFTI YNNMFCAGFH 401 EGGRDSCQGD SGGPHVTEVE GTSFLTGIIS WGEECAMKGK YGIYTKVSRY 451 VNWIKEKTKL TDKTRTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEV 501 TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL 551 HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT 601 KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK 651 LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK

TABLE 19 Polynucleotide sequences of FVII A. Full Length FVII-FcFull Length FVII-Fc DNA Sequence (FYII signal peptideunderlined. Fc region in bold) (SEQ ID NO: 15, which encodesSEQ ID NO: 16)

 201 gctgcggccg ggctccctgg agagggagtg caaggaggag cagtgctcct 251 tcgaggaggc ccgggagatc ttcaaggacg cggagaggac gaagctgttc 301 tggatttctt acagtgatgg ggaccagtgt gcctcaagtc catgccagaa 351 tgggggctcc tgcaaggacc agctccagtc ctatatctgc ttctgcctcc 401 ctgccttcga gggccggaac tgtgagacgc acaaggatga ccagctgatc 451 tgtgtgaacg agaacggcgg ctgtgagcag tactgcagtg accacacggg 501 caccaagcgc tcctgtcggt gccacgaggg gtactctctg ctggcagacg 551 gggtgtcctg cacacccaca gttgaatatc catgtggaaa aatacctatt 601 ctagaaaaaa gaaatgccag caaaccccaa ggccgaattg tggggggcaa 651 ggtgtgcccc aaaggggagt gtccatggca ggtcctgttg ttggtgaatg 701 gagctcagtt gtgtgggggg accctgatca acaccatctg ggtggtctcc 751 gcggcccact gtttcgacaa aatcaagaac tggaggaacc tgatcgcggt 801 gctgggcgag cacgacctca gcgagcacga cggggatgag cagagccggc 851 gggtggcgca ggtcatcatc cccagcacgt acgtcccggg caccaccaac 901 cacgacatcg cgctgctccg cctgcaccag cccgtggtcc tcactgacca 951 tgtggtgccc ctctgcctgc ccgaacggac gttctctgag aggacgctgg1001 ccttcgtgcg cttctcattg gtcagcggct ggggccagct gctggaccgt1051 ggcgccacgg ccctggagct catggtcctc aacgtgcccc ggctgatgac1101 ccaggactgc ctgcagcagt cacggaaggt gggagactcc ccaaatatca1151 cggagtacat gttctgtgcc ggctactcgg atggcagcaa ggactcctgc1201 aagggggaca gtggaggccc acatgccacc cactaccggg gcacgtggta1251 cctgacgggc atcgtcagct ggggccaggg ctgcgcaacc gtgggccact1301 ttggggtgta caccagggtc tcccagtaca tcgagtggct gcaaaagctc1351 atgcgctcag agccacgccc aggagtcctc ctgcgagccc catttcccta1401 ggacaaaact cacacatgcc caccgtgccc agctccagaa ctcctgggcg1451 gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc1501 tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga1551 ccctgaggtc aagttcaact ggtacgtgga cggcgtggag gtgcataatg1601 ccaagacaaa gccgcgggag gagcagtaca acagcacgta ccgtgtggtc1651 agcgtcctca ccgtcctgca ccaggactgg ctgaatggca aggagtacaa1701 gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag aaaaccatct1751 ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgccccca1801 tcccgggatg agctgaccaa gaaccaggtc agcctgacct gcctggtcaa1851 aggcttctat cccagcgaca tcgccgtgga gtgggagagc aatgggcagc1901 cggagaacaa ctacaagacc acgcctcccg tgttggactc cgacggctcc1951 ttcttcctct acagcaagct caccgtggac aagagcaggt ggcagcaggg2001 gaacgtcttc tcatgctccg tgatgcatga ggctctgcac aaccactaca2051 cgcagaagag cctctccctg tctccgggta aa

TABLE 20 Polypeptide Sequences of FVIIFIVII-Fc Monomer Hybrid: created by coexpressing FVII-Fc and Fc chains.A. FVII-Fc chain (signal sequence underlined, Fc region is inbold) (SEQ ID NO: 16) FVII-Fc-Sc Subunit:

 101 WISYSDGDQC ASSPCQNGGS CKDQLQSYIC FCLPAFEGRN CETHKDDQLI 151 CVNENGGCEQ YCSDHTGTKR SCRCHEGYSL LADGVSCTPT VEYPCGKIPI 201 LEKRNASKPQ GRIVGGKVCP KGECPWQVLL LVNGAQLCGG TLINTIWVVS 251 AAHCFDKIKN WRNLIAVLGE HDLSEHDGDE QSRRVAQVIT PSTYVPGTTN 301 HDIALLRLHQ PVVLTDHVVP LCLPERTFSE RTLAFVRFSL VSGWGQLLDR 351 GATALELMVL NVPRLMTQDC LQQSRKVGDS PNITEYMFCA GYSDGSKDSC 401 KGDSGGPHAT HYRGTWYLTG IVSWGQGCAT VGHFGVYTRV SQYIEWLQKL 451 MRSEPRPGVL LRAPFPDKTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS 501 RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS 551 VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPS 601 RDELTKNQVS LTCLVKGFYP SDIAVMWESN GQPENNYKTT PPVLDSDGSF 651 FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK

What is claimed is:
 1. A method of inactivating a virus present duringproduction of a polypeptide of interest, the method comprising thefollowing sequential steps: (a) binding the polypeptide to achromatography matrix, (b) washing the polypeptide-bound chromatographymatrix of step (a) with a first wash solution comprising a salt at a pHabove 6.5, (c) washing the polypeptide-bound chromatography matrix ofstep (b) with a second wash solution comprising a salt at aconcentration greater than about 0.5 M and a pH of lower than about 4.0,and (d) washing the polypeptide-bound chromatography matrix of step (c)with a third wash solution comprising a salt at a pH above 6.5, suchthat virus present during production of the polypeptide is inactivated,wherein the polypeptide comprises a recombinant FcRn binding partner(FcRn BP).
 2. The method of claim 1, wherein the chromatography matrixis an affinity chromatography matrix.
 3. The method of claim 2, whereinthe affinity chromatography matrix is a Protein A column.
 4. The methodof claim 3, wherein the Protein A column comprises a Protein A ligand,which is immobilized on a matrix comprising a dextran based matrix,agarose based matrix, polystyrene based matrix, hydrophilic polyvinylethyl based matrix, rigid polymethacrylate based matrix, porous polymerbased matrix, controlled pore glass based matrix, or any combinationthereof.
 5. The method of claim 1, wherein the chromatography matrix isa mixed-mode chromatography matrix.
 6. The method of claim 5, whereinthe mixed-mode chromatography matrix comprises dextran based matrix,agarose based matrix, polystyrene based matrix, polyvinyl ethylhydrophilic polymer based matrix, macroporous highly crosslinked polymerbased matrix, hydroxyapatite ((Ca₅(PO₄)₃OH)₂) based matrix,fluoroapatite ((Ca₅(PO₄)₃F)₂) based matrix, or any combinations thereof.7. The method of claim 1, wherein the salt reduces an elution of thepolypeptide during step (c) to less than 30%.
 8. The method of claim 7,wherein the salt reduces the elution of the polypeptide during step (c)to less than 20%.
 9. The method of claim 1, wherein the pH of the secondwash solution is about 2.5 to about 4.0.
 10. The method of claim 9,wherein the pH of the second wash solution is about 3.0.
 11. The methodof claim 1, wherein the salt is a sodium salt, a potassium salt, or anammonium salt.
 12. The method of claim 1, wherein the second washsolution further comprises one or more components selected from thegroup consisting of a polymer, an organic solvent, a detergent,arginine, an arginine derivative, and any combination thereof.
 13. Themethod of claim 1, wherein the wash solution further comprises PEG,ethanol, acetone, or octylphenol ethylene oxide condensate.
 14. Themethod of claim 1, wherein the polypeptide is bound to thechromatography matrix at a pH from about 6.0 to about 8.0.
 15. Themethod of claim 1, wherein the method further comprises eluting thepolypeptide from the chromatography matrix with an elution solution. 16.The method of claim 1, wherein the polypeptide further comprises aclotting factor comprising Factor V (FV), Factor VII (FVII), Factor VIII(FVIII), Factor IX (FIX), Factor X (FX), Factor XI (FXI), von WillebrandFactor (VWF), or any combinations thereof.
 17. The method of claim 1,wherein the FcRn BP comprises Fc.
 18. The method of claim 1, wherein thesalt is at a concentration between about 0.5M to about 2.5M.
 19. Themethod of claim 18, wherein the salt is at a concentration between about0.5M and about 2.0M.
 20. The method of claim 18, wherein the salt is ata concentration between about 1.0M and about 2.0M.
 21. The method ofclaim 18, wherein the salt is at a concentration between about 1.5M and2.0M.
 22. A method of inactivating a virus present during production ofa polypeptide of interest, comprising: (a) binding the polypeptide to achromatography matrix, and (b) washing the polypeptide-boundchromatography matrix with a wash solution comprising a salt at aconcentration greater than about 0.5M and a pH of about 3.0 to about4.0, wherein the polypeptide of interest is a Factor IX (FIX) fusionprotein comprising a FIX polypeptide and a Fc region (rFIXFc), whereinthe FIX polypeptide is a recombinant FIX covalently linked to thedimeric Fc region of immunoglobulin G1 (IgG1) with no interveningsequence, and wherein the polypeptide of interest comprises an aminoacid sequence according to amino acids 47 to 687 of SEQ ID NO:
 14. 23.The method of claim 22, wherein the chromatography matrix is an affinitychromatography matrix.
 24. The method of claim 22, wherein thechromatography matrix is a Protein A column.
 25. The method of claim 22,wherein the chromatography matrix is a Protein A column comprising aProtein A ligand, which is immobilized on a matrix comprising a dextranbased matrix, agarose based matrix, polystyrene based matrix,hydrophilic polyvinyl ethyl based matrix, rigid polymethacrylate basedmatrix, porous polymer based matrix, controlled pore glass based matrix,or any combination thereof.
 26. The method of claim 22, wherein thechromatography matrix is a mixed-mode chromatography matrix.
 27. Themethod of claim 22, wherein the chromatography matrix is a mixed-modechromatography matrix comprising dextran based matrix, agarose basedmatrix, polystyrene based matrix, polyvinyl ethyl hydrophilic polymerbased matrix, macroporous highly crosslinked polymer based matrix,hydroxyapatite ((Ca₅(PO₄)₃OH)₂) based matrix, fluoroapatite((Ca₅(PO₄)₃F)₂) based matrix, or any combinations thereof.
 28. Themethod of claim 22, wherein the salt is a sodium salt, a potassium salt,or an ammonium salt.
 29. The method of claim 22, wherein the washsolution further comprises one or more components selected from thegroup consisting of a polymer, an organic solvent, a detergent,arginine, an arginine derivative, and any combination thereof.
 30. Themethod of claim 22, wherein the wash solution further comprises PEG,ethanol, acetone, or octyphcnol octylphenol ethylene oxide condensate.31. The method of claim 22, wherein the method further comprises elutingthe polypeptide from the chromatography matrix with an elution solution.32. The method of claim 22, wherein the salt is NaCl.
 33. The method ofclaim 22, wherein the salt is sodium phosphate.
 34. The method of claim22, wherein the salt is ammonium sulfate.
 35. The method of claim 22,wherein the salt is at a concentration between about 0.5M to about 2.5M.36. The method of claim 35, wherein the salt is at a concentrationbetween about 0.5M and about 2.0M.
 37. The method of claim 35, whereinthe salt is at a concentration between about 1.0M and about 2.0M. 38.The method of claim 35, wherein the salt is at a concentration betweenabout 1.5M and 2.0M.
 39. The method of claim 22, wherein the salt is ata pH of about 3.4.
 40. The method of claim 22, wherein the salt is at apH of about 3.5.
 41. The method of claim 22, wherein the salt is at a pHof about 3.6.